Method for reducing the immune response to a biologically active protein

ABSTRACT

A new use of a molecule comprising at least one moiety which is a biologically active protein and at least one moiety capable of binding to a serum albumin of a mammal is provided, for preparation of a medicament which elicits no or a reduced immune response upon administration to the mammal, as compared to the immune response elicited upon administration to the mammal of the biologically active protein per se. Also provided is a method of reducing or eliminating the immune response elicited upon administration of a biologically active protein to a human or non-human mammal, which comprises coupling the polypeptide to at least one moiety capable of binding to a serum albumin of the mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.11/547,888, filed Nov. 15, 2007, now U.S. Pat. No. 8,642,743 issued Feb.4, 2014, which is a National Stage of International Patent ApplicationNo. PCT/GB2005/001321, filed Apr. 6, 2005, which claims priority toEuropean Patent Application No. 04008299.2, filed Apr. 6, 2004. Theseapplications are incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to methods and uses that reduce theimmunogenicity of biologically active proteins. In particular, itrelates to the use of a molecule comprising a biologically activeprotein part and an albumin binding part for preparation of amedicament.

BACKGROUND OF THE INVENTION Serum Albumin

Serum albumin is the most abundant protein in mammalian sera (40 g/l;≈0.7 mM in humans), and one of its functions is to bind molecules suchas lipids and bilirubin (Peters T, Advances in Protein Chemistry 37:161,1985). The half-life of serum albumin is directly proportional to thesize of the animal, where for example human serum albumin (HSA) has ahalf-life of 19 days and rabbit serum albumin has a half-life of about 5days (McCurdy T R et al, J Lab Clin Med 143:115, 2004). Human serumalbumin is widely distributed throughout the body, in particular in theintestinal and blood compartments where it is mainly involved in themaintenance of osmolarity. Structurally, albumins are single-chainproteins comprising three homologous domains and totaling 584 or 585amino acids (Dugaiczyk L et al, Proc Natl Acad Sci USA 79:71 (1982)).Albumins contain 17 disulfide bridges and a single reactive thiol,Cys34, but lack N-linked and O-linked carbohydrate moieties (Peters,1985, supra; Nicholson J P et al, Br J Anaesth 85:599 (2000)). The lackof glycosylation simplifies recombinant expression of albumin. Thisproperty, together with the fact that the three-dimensional structure isknown (He X M and Carter D C, Nature 358:209 (1992)), has made it anattractive candidate for use in recombinant fusion proteins. Such fusionproteins generally combine a therapeutic protein (which would be rapidlycleared from the body upon administration of the protein per se) and aplasma protein (which exhibits a natural slow clearance) in a singlepolypeptide chain (Sheffield W P, Curr Drug Targets Cardiovacs HaematolDisord 1:1 (2001)). Such fusion proteins may provide clinical benefitsin requiring less frequent injection and higher levels of therapeuticprotein in vivo.

Fusion or Association with HSA Results in Increased In Vivo Half-Life ofProteins

Serum albumin is devoid of any enzymatic or immunological function and,thus, should not exhibit undesired side effects upon coupling to abioactive polypeptide. Furthermore, HSA is a natural carrier involved inthe endogenous transport and delivery of numerous natural as well astherapeutic molecules (Sellers E M and Koch-Weser M D, AlbuminStructure, Function and Uses, eds Rosenoer V M et al, (Pergamon, Oxford,p 159 (1977)). Several strategies have been reported to eithercovalently couple proteins directly to serum albumins or to a peptide orprotein that will allow in vivo association to serum albumins. Examplesof the latter approach have been described e.g. in EP 486 525 and U.S.Pat. No. 6,267,964, in WO01/45746 and in Dennis et al, J Biol Chem277:35035-43 (2002). The first two documents describe inter alia the useof albumin-binding peptides or proteins derived from streptococcalprotein G (SpG) for increasing the half-life of other proteins. The ideais to fuse the bacterially derived, albumin-binding peptide/protein to atherapeutically interesting peptide/protein, which has been shown tohave a rapid clearance in blood. The thus generated fusion protein bindsto serum albumin in vivo, and benefits from its longer half-life, whichincreases the net half-life of the fused therapeutically interestingpeptide/protein. WO01/45746 and Dennis et al relate to the same concept,but here, the authors utilize relatively short peptides to bind serumalbumin. The peptides were selected from a phage displayed peptidelibrary. In Dennis et al, earlier work is mentioned in which theenhancement of an immunological response to a recombinant fusion of thealbumin binding domain of streptococcal protein G to human complementreceptor Type 1 was found. US Patent application no.2004/0001827(Dennis) also discloses the use of constructs comprisingpeptide ligands, again identified by phage display technology, whichbind to serum albumin conjugated to bioactive compounds for tumourtargeting. Whilst the constructs are said to have improvedpharmacokinetic and pharmacodynamic properties, there is no disclosureor suggestion in this document of a reduction in the immunogenicity ofthe constructs compared to the unconjugated bioactive compounds. Thereis no suggestion that further serum albumin-binding conjugate moleculeswould be desirable.

As an alternative, the therapeutically interesting peptide/protein inquestion may also be fused directly to serum albumin, as mentioned aboveand described by Yeh et al (Proc Natl Acad Sci USA 89:1904 (1992)) andSung et al (J Interferon Cytokine Res 23:25 (2003)). Yeh et al describethe conjugation of two extracellular Ig-like domains (V1 and V2) of CD4to HSA. The HSA-CD4 conjugate has a retained biological activity of CD4,but the half-life was reported to increase 140 times in an experimentalrabbit model, as compared to CD4 alone. Soluble CD4 alone has anelimination half-life of 0.25±0.1 hrs, whereas the elimination half-lifeof HSA-CD4 was reported to be 34±4 hrs. A prolonged eliminationhalf-life was also observed for interferon-beta (IFN-β) upon conjugationto HSA, as outlined by Sung et al, supra. Here, IFN-β-HSA conjugate wasevaluated in primates, and the half-life of IFN-β was reported toincrease from 8 hrs alone to 36 to 40 hrs when conjugated to HSA.

Albumin Binding Domains of Streptococcal Protein G

Streptococcal protein G (SpG) is a bifunctional receptor present on thesurface of certain strains of streptococci and is capable of binding toboth IgG and serum albumin (Björck L et al, Mol Immunol 24:1113 (1987)).The structure is highly repetitive with several structurally andfunctionally different domains (Guss B et al, EMBO J. 5:1567 (1986)).More precisely, SpG comprises one Ig-binding motif and three serumalbumin binding motifs (Olsson A et al, Eur J Biochem 168:319 (1987)).

The albumin-binding protein BB, derived from streptococcal protein G,has 214 amino acid residues and contains about 2.5 of thealbumin-binding motifs of SpG (Nygren P-Å et al, J Mol Recognit 1:69(1988)). It has been shown previously that BB has several propertiesthat make it highly suitable as a fusion partner for peptide immunogenswith the purpose of creating powerful vaccines. For example, BB has beenfused with repeated structures from the P. falciparum malaria antigenPf155/RESA (M3) (Sj öander A et al, J Immunol Meth 201:115 (1997)), andto a respiratory syncital virus (RSV) (Long) G protein fragment (G2Na)(Power U F et al, Virol 230:155 (1997)). Both BB-M3 and BB-G2Na wereable to trigger strong and long-lasting antibody responses against bothimmunogen fusion moieties in several animal models. BB-M3 induced hightitres of antibodies in rabbits after covalent conjugation to immunestimulating complexes (iscoms) (Sjoöander A et al, Immunometh 2:79(1993)) and is immunogenic in mice (Sjoöander et al, 1997, supra) andAotus monkeys (Berzins K et al, Vaccine Res 4:121 (1995)). The observedeffect was observed in the presence of a potent adjuvant, e.g. Freund'scomplete adjuvant (FCA). Furthermore, BB-G2Na induced detectable andprotective antibody responses in both mice and man (Power et al, 1997,supra; Power U F et al, J Infect Dis 184:1456 (2001)). In agreement withBB-M3, this effect was observed in the presence of a strong adjuvant, inthis case mannitol and aluminum phosphate.

The structure of one of the serum albumin-binding motifs of SpG,designated A3, ABD3 or just ABD (“albumin binding domain”), has beendetermined (Kraulis P J et al, FEBS Lett 378:190 (1996)). This studyrevealed a three-helix bundle domain, surprisingly similar in structureto the Ig binding domains of staphylococcal protein A. The SpG domainABD corresponds to 46 amino acids.

The albumin binding parts of SpG have been epitope mapped closely, asdescribed by Goetsch et al (Clin Diagn Lab Immunol 10:125 (2003)).

Other Albumin-Binding Domains

Albumin-binding proteins are found in other bacteria. For example,naturally occurring albumin-binding proteins include certain surfaceproteins from Gram⁺ bacteria, such as streptococcal M proteins (e.g.M1/Emm1, M3/Emm3, M12/Emm12, EmmL55/Emm55, Emm49/EmmL49 and Protein H),streptococcal proteins G, MAG and ZAG, and PPL and PAB from certainstrains of Finegoldia magna (formerly Peptostreptococcus magnus). Seereview of Gram⁺ surface proteins by Navarre W W and Schneewind O(Microbiol Mol Biol Rev 63:174-229 (1999)), and references containedtherein. The characteristics of albumin-binding by some of theseproteins have been elucidated further, by e.g. Johansson M U et al (JBiol Chem 277:8114-8120 (2002)); Linhult et al (Prot Sci 11:206-213(2002), and Lejon S. et al J. Biol. Chem. 279, 41, 2004, 42924-42928).

Clinical Implications of Immunogenicity

Most biologically active proteins, including proteins that are more orless identical to proteins native to the species in question, induceantibody responses upon administration to a significant fraction ofsubjects. The main factors that contribute to immunogenicity arepresence of foreign epitopes, e.g. new idiotopes, different Ig allotypesor non-self sequences, impurities and presence of protein aggregates. Inthe majority of cases, the induced antibodies have no biological orclinical effects. Where a clinical effect is observed, the most commonis a loss of efficacy of the biopharmaceutical.

However, cases with more serious adverse events have been reported. Onthese occasions, antibodies raised against a protein pharmaceuticalcross-reacted with endogenous proteins. Erythropoietin is such anexample. When administering erythropoietin to humans, immune responseswere induced that led to pure red cell aplasia in the patients(Casadevall N et al, New Eng J Med 346:469 (2002)). The specificantibodies that were generated were of high affinity, and were alsoshown to cross-react with other forms of erythropoietin such as Eprex®,Epogen® and NeoRecormon®, which indicates that the reactivity was mostlikely directed against the erythropoietin active site conformation.

Another example is thrombopoietin, which, upon administration to humans,resulted in the production of neutralizing antibodies. The antibodiesinhibited the activity of endogenous thrombopoietin, which resulted inautoimmune thrombocytopenia (Koren E et al, Curr Pharm Biotech 3:349(2002)).

Given that the clinical use of biopharmaceuticals often elicits animmune response, immunogenicity is a risk factor to be managed duringthe development of all biopharmaceutical products. Besideserythropoietin and thrombopoietin mentioned above, several otherbiopharmaceuticals have also been reported to induce immune responses inpatients. Examples are ciliary neurotrophic factor (CNTF),granulocyte-macrophage colony-stimulating factor (GM-CSF), growthhormone (GH), insulin and interferon-beta (IFN-β). The reasons why theabove proteins are observed to generate antibodies in the treatedpatients differ depending on the product. More precisely, the mainfactor in the immunogenicity of insulin appeared to have been proteinimpurities which acted as adjuvants, whereas in the case of IFN-β themain factors were believed to be lack of glycosylation when the proteinwas produced in a bacterial host cell and presence of aggregates due tolow solubility (Karpusas M et al, Cell Mol Life Sci 54:1203 (1998)).Before the advent of recombinant human GH, GH from human cadavers wasused to treat GH-deficiency in children. Mainly due to a high content ofprotein impurities, 45% of the children produced antibodies against thisfirst generation of products (Raben M S, Recent Prog Horm Res 15:71(1959)). When recombinant GH, which includes an extra methionine residuethat enables production in E. coli, was administered to the patients,the incident of antibodies decreased to 8.5% (Okada Y et al, EndocrinolJpn 34:621 (1987)). The immunogenicity of GH is more complex thanpercent identity to the self-protein or lack of immune tolerance,judging from the fact that only one twin developed antibodies torecombinant GH when twins with homozygous GH deletion mutants weretreated with the therapeutic protein (Hauffa B P et al, Acta Endocrinol121:609 (1989)). Recombinant human CNTF was produced with the purpose oftreating patients with amyotrophic lateral sclerosis (ALS), since CNTFis believed to enhance the survival of motor neurons. Unfortunately,more than 90% of these patients were tested positive for anti-CNTFantibodies after two weeks of treatment. The clinical effect of thetherapeutic protein was severely hampered by the specific antibodies, asshown by the ALS CNTF Study Group report in 1995 (Clin Neuropharmacol18:515 (1995)). A second generation of CNTF, which contains a truncatedC-terminus and is PEGylated, is currently under development.

Furthermore, the immunogenicity of therapeutic antibody molecules is asignificant problem which severely limits their widespread and repeatedapplication in treating many diseases.

Different Strategies to Decrease Immunogenicity

Technologies that reduce immunogenicity of proteins are thereforeneeded. Actually, the importance of such technologies is increasing,since it is becoming more and more common that protein pharmaceuticalshave amino acid sequence modifications compared to the naturallyoccurring protein, or are altogether comprised of amino acid sequencesforeign to the subject. One important method to prevent immunogenicityis by optimization of production, purification and formulation of thebiopharmaceutical protein to generate soluble, non-aggregated, nativeprotein which is free of contaminating adjuvants. There are severalreports on the reduction of immunogenicity of proteins, e.g. humangrowth hormone (Moore W V et al, J Clin Endocrin Meth 51:691 (1980)) andinterferon-α2a (Hochuli E, J Inter Cyto Res 17:15 (1997)), throughimprovement of purification and formulation.

Other methods to alter immunogenicity are directed against the actualsequence or structure of the protein in question, and sometimes referredto as “deimmunization methods”. Examples of such strategies are epitopeneutralization, gene-shuffling, chemical modifications and immunetolerance. Epitope neutralization involves rational identification ofdominant T and/or B-cell epitopes using in silico and/or in vitromethods, and subsequent redesign of highlighted sequences to eliminatethe dominant epitopes and, hopefully, obtain decreased immunogenicity(Stickler M M et al, J Immunother 6:654 (2000); US Patent ApplicationPublication No. 2003/0166877). Another example of deimmunization andgene-shuffling is the humanization of antibody molecules (Kuus-Reichel Ket al, Clin Diagn Lab Immunol 1:365 (1994)). The immunogenicity has beenreported to drop going from murine to chimeric to fully humanantibodies. The evolution of human proteins using DNA gene-shufflinginvolves homologydependent recombination of DNA fragments to generateordered chimaeras of genes. Gene-shuffling could be useful when seekingproteins with reduced immunogenicity and with retained biologicalactivity (Pavlinkova G et al, Int. J. Cancer 94:717 (2001)).

Another method to alter the antigenicity (binding to pre-existingantibodies) and immunogenicity (ability to induce new immune responses)of a protein is to modify the protein chemically. Chemical modificationscan be accomplished using covalently bound polymers such as polyethyleneglycol (PEG) (Molineux G, Pharmacother 23:3 (2003)) and Dextran(Kobayashi K et al, J Agric Food Chem 49:823 (2001)), or performingneutralization of positive charges with succinic anhydride. PEG is anon-toxic, highly soluble molecule that has been shown to increase thehalf-life in vivo of proteins covalently bound thereto, and to reducethe immunogenicity of such proteins (Molineux G, supra). The PEGapproach is commonly referred to as “PEGylation” of a protein.

Induction of immune tolerance offers a more acceptable means forpreventing an immune response than PEGylation, since no chemicaladditions are made to the therapeutic molecule. The same pharmaceuticalis administered, which e.g. ensures a better patient compliance. Thisapproach has been tried for example in the context of administration offactor VIII to patients suffering from hemophilia A. One complication inusing factor VIII to treat hemophilia A is the generation of inhibitoryantibodies to the therapeutic protein, which is observed in aboutone-third of all patients (Scharrer I, Haemophilia 5:253 (1999)). Dailyinjections of large doses of factor VIII together with immunosuppressiveagents, which should be given for time periods of from months to yearsis one strategy that is being pursued in the effort of trying to limitthe immune response.

Drawbacks of Current Strategies for Reducing Immunogenicity

The drawbacks of the different approaches to reduce immunogenicity areseveral. For one thing, it is difficult to achieve covalent attachmentto a protein of PEG molecules without obstructing the active sites thatare essential for drug efficacy. Avoiding this is a major challenge inPEGylation. There is a great variation in the quality of PEGylatedproducts, and numerous factors have shown to play a part in thisvariation: the presence or absence of linkers between PEG and theprotein; the nature and stability of the bond(s) between the PEG, linkerand protein; the impact of PEG attachment on surface charge of theresulting PEGylated protein; the coupling conditions; the requirement ofproving that the product is homogeneous; and the relative toxicity ofthe activated polymer. Moreover, considerable modifications of theprototype method, and also a process of biological optimization havebeen required to achieve good results in terms of conservation ofbioactivity. Any reduction in activity has to be addressed by increasingthe treatment dosage, which once again increases the risk of an immunereaction to those molecules. Another drawback of the PEGylation approachis that PEGylated therapeutics increase the cost of treatment by anestimated USD 1000 per month per patient. There has been very littlesuccess with polymers other than PEG with regard to improving thepharmacological and immunological properties of therapeutic proteinmolecules (Burnham N L, Am J Hosp Pharm 51:210 (1994)).

As stated above, the immunogenicity of therapeutic antibody moleculeshas been addressed using the humanization approach. Humanization hasworked well for some murine antibodies, e.g. HERCEPTIN®, which isapproved for treatment of some breast cancers. In other cases, humanizedantibodies, e.g. CAMPATH®-1H used for treatment of rheumatoid arthritis,still induce an immune response in 60% of the treated patients.Additionally, data from animal studies have shown that rodents areclearly not tolerant of antibodies from the same species and strain(Cobbold S P et al, Meth Enzym 127:19 (1990)) and fully human antibodiesare believed to have the potential to evoke anti-idiotypic antibodiesjust like any other antibody.

Furthermore, the “deimmunization” approach, for example the targetedelimination of T and B-cell epitopes, is not as trivial as it may seem.The algorithms that are available for in silico prediction of epitopesmay not be reliable. In the case of predicting B-cell epitopes, this isfairly difficult to do using algorithms, since such epitopes to a greatextent are conformational epitopes. T-cell epitopes, on the other hand,are linear, which means that the existing in silico tools are morereliable. Unfortunately, most algorithms are suitable for identifyingmajor histocompatibility complex (MHC) class I associated peptides andnot MHC class II associated peptides. Since the latter are more relevantfor T-helper cell activation, this is a drawback when seeking to reduceantibody responses. Furthermore, the great polymorphism of MHC moleculesmakes it difficult to predict, using immunoinformatics, the majority ofthe T-cell epitopes of any given protein antigen. It is important toremember that epitopes identified by immunoinformatics should always beverified by experimental studies in e.g. in vitro human T-cellstimulation assays. One of the reasons for this is that binding of animmunogenic peptide (i.e. a T-cell epitope) to MHC class II molecules isnot sufficient to ensure recognition by a given T cell antigen receptorthat has specificity for the peptide. The studies that are necessary forthe identification of both T and B-cell epitopes are time-consuming aswell as experimentally difficult.

The major disadvantages involved in inducing tolerance to differenttherapeutics, such as factor VIII exemplified above, are the effects oflong-term treatment with immunosuppressive agents (such as sensitivityto infections following suppression of the immune system, and potentialtoxic effects of the agents) and the high cost involved. It has beenestimated that the induction of immune tolerance against factor VIII ina paediatric patient costs nearly USD 1 million.

Despite the existence of strategies for reducing the immunogenicity ofbiopharmaceuticals and other proteins with biological activity, none ofthese strategies has proved itself useful in all situations where thereduction or elimination of immunogenicity is desired. Thus, there is acontinued need for complementary approaches to the problem.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to meet this need, through theprovision of a new way to avoid problems associated with immunogenicityof proteins upon administration thereof to a mammalian subject.

It is a related object of the present invention to reduce, or ideallycompletely avoid, the generation of antibodies against an administeredbiopharmaceutical or protein drug.

It is another object of the present invention to exploit the abundanceof serum albumin in the mammalian blood for a novel purpose.

In view of these objects, and others which are apparent to the skilledperson through the disclosure herein, the present invention, in itsvarious aspects, provides a surprising alternative to the hitherto knownalternatives for solving the immunogenicity problem.

Thus, in one aspect, the invention provides the use of a molecule (“themolecule of the invention”) comprising at least one moiety which is abiologically active protein and at least one moiety capable of bindingto a serum albumin of a mammal, for the preparation of a medicamentwhich elicits no or a reduced immune response upon administration to themammal, as compared to the immune response elicited upon administrationto the mammal of the biologically active protein per se.

In another aspect, the invention provides a method of reducing oreliminating the immune response elicited upon administration of abiologically active protein to a human or non-human mammal, whichcomprises coupling the polypeptide to at least one moiety capable ofbinding to a serum albumin of the mammal to form a molecule prior toadministration of the molecule (“the molecule of the invention”) to themammal.

In still another aspect, the invention provides an improvement in amethod of administering a biologically active protein to a non-human orhuman mammal, wherein the improvement comprises administering a moleculecomprising at least one moiety which is derived from the biologicallyactive protein and at least one moiety capable of binding to a serumalbumin of a mammal (“the molecule of the invention”), whereby themolecule elicits no or a reduced immune response as compared to acompound consisting of the biologically active protein per se.

In another aspect of the invention, there is provided a moleculecomprising at least one moiety which is a biologically active proteinand at least one moiety capable of binding to a serum albumin of amammal (“the molecule of the invention”), which elicits no or a reducedimmune response upon administration to the mammal, as compared to theimmune response elicited upon administration to the mammal of thebiologically active protein per se.

The invention also provides a composition comprising such a molecule anda pharmaceutically acceptable carrier, and the use of such a molecule inthe preparation of a medicament.

As a basis for the different aspects of the invention, it has beensurprisingly shown by the present inventors that it is feasible toreduce substantially or even eliminate the immune response against abiologically active protein through covalent coupling of thebiologically active protein to a moiety with affinity for a serumalbumin. The immune response elicited by administration of the resultingmolecule to a mammal is significantly reduced in comparison with thatelicited by administration of the biologically active protein without analbumin-binding moiety covalently coupled thereto. This is a surprisingobservation indeed, especially in view of the fact that the onlyimmunological effect shown in the prior art of coupling a biologicallyactive protein or peptide to an albumin-binding moiety is an increase inthe generation of antibodies to an immunogen coupled to thealbumin-binding protein BB in the presence of an adjuvant (seeBackground of the invention).

In one embodiment of the invention, the immune response that is reducedor eliminated is a humoral immune response. The humoral immune responsereduced or eliminated in this embodiment may be for example theproduction of antibodies especially of the IgG isotype.

Without wishing to be bound by any specific theory, it is thought thatassociation of the molecule with serum albumin within the body afteradministration thereof to the subject mammal results in the immunesystem of the subject ignoring the molecule, so that an immune responseto the molecule does not come about. The equilibrium of albuminassociation is likely to be shifted very far in the direction of thecomplex between the molecule and albumin.

The moiety capable of binding to a serum albumin of a mammal may be aprotein or peptide, such as a polypeptide or oligopeptide.

The moiety capable of binding to a serum albumin of a mammal may be anaturally occurring polypeptide with the desired ability to interactwith a serum albumin. Also, fragments or derivatives of such naturallyoccurring polypeptides may be used as the albumin-binding moiety,provided of course that the capability of binding albumin is at leastpartially retained in such a fragment or derivative. As non-limitingexamples of naturally occurring polypeptides with albumin-bindingactivity, mention can be made of certain surface proteins from Gram⁺bacteria, such as streptococcal M proteins (e.g. M1/Emm1, M3/Emm3,M12/Emm12, EmmL55/Emm55, Emm49/EmmL49 and Protein H), streptococcalproteins G, MAG and ZAG, and PPL and PAB from certain strains ofFinegoldia magna (formerly Peptostreptococcus magnus). See review ofGram⁺ surface proteins by Navarre W W and Schneewind O (Microbiol MolBiol Rev 63:174-229 (1999)), and references contained therein. Thecharacteristics of albumin-binding by some of these proteins have beenelucidated further, by e.g. Johansson M U et al (J Biol Chem277:8114-8120 (2002)); Linhult et al (Prot Sci 11:206-213 (2002), andLejon S. et al J. Biol. Chem. 279, 41, 2004, 42924-42928). With theknowledge from these and other publications of what domains areresponsible for albumin-binding in these and other proteins, it lieswithin the normal capability of the person skilled in the field to finda suitable fragment or derivative of any of the listed proteins for useas the albumin-binding moiety in the molecule to be used in the contextof the present invention. For example, Protein PAB contains a 53 aminoacid residue albumin-binding domain, known as the GA module, which hashigh sequence homology with the streptococcal ABD is discussed in detailin Lejon et al supra. In particular, this paper discloses theidentification of amino acid residues which are important in the bindingof the module with human serum albumin. The skilled worker can produceserum albumin-binding moieties having suitable residues which cause theserum albumin-binding moiety to bind to an albumin or to enhance thealbumin binding properties of the moiety.

Specifically Lejon et al supra found that the hydrophobic core of theinterface between human serum albumin and the GA module is lined withresidues Phe-228, Ala-229, Ala-322, Val-325, Phe-326, and Met-329 fromhuman serum albumin, and residues Phe-27, Ala 31, Leu-44, and Ile-48from GA. The skilled worker may provide a serum-albumin binding moietythat includes alternative amino acid residues which contribute to thisinteraction surface, e.g. to the hydrophobic core of the interface, orwhich contribute to the surrounding hydrogen bond interactions so as toenhance binding of the molecule to albumin. Such variants havingretained or enhanced affinity for serum-albumin may be constructed basedon the information on the complex found in Lejon et al. supra, andcomprise alternative surface residues of helix 2 and 3 and of the loopspreceding and following helix 2. In addition to altering the bindingsurface of the crystallized complex by replacing residues directlyinvolved in binding, alternative amino acids may be replaced as a meansof obtaining an enhanced binding affinity for serum-albumin due toindirect structural effects or electrostatic steering forces (Low etal., J. Mol. Biol. 260: 359-368, 1996; Schreiber and Fersht, Struct.Biol. 3: 427-431, 1996), or as a means of introducing simultaneousinteractions with other portions of serum-albumin. It will beappreciated that the interactions may be in the nature of hydrogenbonds, van der Waals interactions or electrostatic bonds according tothe context. All sequence variants resulting from either or both ofthese approaches for modification are considered as directly relatedvariants of the serum-albumin binding domain described by Kraulis et alsupra and used in the examples in this application.

The possible formation of a complex with a fatty acid at the bindinginterface between human serum albumin is discussed by Lejon et al supra.The applications of the present invention may be modified to enhancebinding in the presence or the absence of a fatty acid.

Thus, as stated above and in the Background section, one naturallyoccurring polypeptide with an albumin-binding function is streptococcalprotein G, SpG. Intact SpG, or any albumin-binding domain or fragment orderivative thereof, may therefore be used as the albumin-binding moietyin the molecule used in accordance with the present invention. Oneexample of such a domain with albumin-binding capability is the SpGdomain ABD (the 46 amino acid domain also referred to in the prior artas ABD3 or A3. See e.g. Kraulis P J et al, supra). Variants or fragmentsthereof with retained albumin-binding capacity may of course also beuseful. For example, the preferred binding affinity for albumin of asuitable variant or fragment may be as set out below.

As another non-limiting alternative, the moiety capable of binding to aserum albumin of a mammal may be an albumin-binding peptide having fromabout 5 to about 40 amino acid residues, such as from about 10 to about20 amino acid residues. Such peptides have been described e.g. inWO01/45746 and in Dennis et al, J Biol Chem 277:35035-43 (2002) in theapplication of prolonging the half-life of a biologically activeprotein. In particular, peptides comprising the amino acid sequenceDICLPRWGCLW (SEQ ID NO:1) and/or peptides comprising the amino acidsequence DLCLRDWGCLW (SEQ ID NO:2) and/or peptides comprising the aminoacid sequence DICLARWGCLW (SEQ ID NO:3) or albumin-binding derivativesof those sequences may be useful as the albumin-binding moiety in thecontext of the present invention. Specific examples of usefulalbumin-binding peptides are found in Tables II, III and IV of Dennis etal, supra, and on p 12-13 of WO01/45746, these sections being herebyincorporated by reference into the present disclosure.

The moiety capable of binding to a serum albumin of a mammal may,alternatively, be an organic, non-proteinaceous compound with affinityfor the mammalian serum albumin. The moiety is preferably a radical ofsuch an organic compound, which is covalently bound to the biologicallyactive protein moiety. Compounds with affinity for serum albumin areknown in the art, and may for example be selected from the groupconsisting of non-steroidal anti-inflammatory drugs (NSAIDs), such asibuprofen and carprofen; diclofenac; salicylic acid; warfarin; propofol;and halothane.

In order to obtain an efficient association of the molecule with theserum albumin of the mammal to which it is administered the moleculeshould have a binding affinity for the albumin such that the K_(D) ofthe interaction is ≦10⁻⁶ M, such as ≦10⁻⁷ M≦10⁻⁸ M, ≦10⁻⁹ M, ≦10⁻¹⁰ M,≦10⁻¹¹ M, ≦10⁻¹² M, ≦10⁻¹³ M or ≦10⁻¹⁴ M. However, in certaincircumstances it may be desirable that the albumin binding is notexcessively tight, so that the molecule used in accordance with theinvention is able to dissociate and perform its intended function in thebody. The K_(D) of a biospecific interaction, such as the one betweenalbumin and the molecule in the present invention, may for example bemeasured using surface plasmon resonance as known to the skilled person,using for example a Biacore® instrument.

The albumin-binding moiety of the molecule of the invention, andtherefore the molecule itself, has an affinity for a serum albumin froma certain mammal. Suitably, the subject to which the molecule isadministered belongs to the same mammalian species, so that theassociation of the albumin-binding moiety and the albumin in the serumof the subject is optimal. Preferably, the albumin-binding moiety isadapted to enhance binding to the serum albumin of a certain mammal. Forexample, where the mammal is a monkey, the albumin-binding moiety mayhave enhanced affinity for simian serum albumin. Similarly in the caseof a human subject the albumin binding moiety may be modified to enhancethe affinity of the molecule of the invention for human serum albumin.

The affinity of the moiety capable of binding to a serum albumin of amammal may be modified to suit the mammal to be treated. At any timethere may be a small amount of the molecule of the invention not boundto albumin. In pharmacokinetic terminology the total exposure over timeof non-bound molecule may be expressed as the area under the curve(AUC). It is appreciated by a person within the field ofpharmacokinetics that the value of this AUC in a given species willdepend both on the affinity to albumin and on the half-life of albumin.In particular, the affinity of the moiety capable of binding to a serumalbumin of a mammal, and therefore that of the molecule of theinvention, may be arranged so that it is greater for use in mammals suchas humans where the half life of the serum albumin is greater. Thus, ahigher affinity may be required in a species having a longer circulationtime of albumin, such as in a human individual. For example, theaffinity of the moiety capable of binding to a serum albumin may beincreased by mutating that moiety. Current experiments in the mouseconfirm the hypothesis that the AUC should be minimized to avoid animmune response; a mutant having a too low affinity did not yieldprotection against an immune response. Thus, a higher affinity may berequired in a species having a longer circulation time of albumin, suchas in a human individual. Therefore, the affinity towards human serumalbumin may preferably be increased by mutating the albumin-bindingmoiety as discussed above.

However, it is possible for one and the same albumin-binding moiety tobe capable of binding to serum albumin from different mammalian species.Thus, for example, SpG and its fragments are capable of binding to serumalbumins from at least mouse, rat and human beings. In one embodiment ofthe invention, the reduction or elimination of immune response accordingto the invention is achieved upon administration of the molecule to ahuman being. In other embodiments, the effect is exploited in connectionwith administration to another, non-human, mammal.

The molecule which elicits a reduced immune response also comprises abiologically active protein moiety. The biologically active proteinmoiety may be any protein that one wishes to administer to a mammal fora given purpose, for example for a therapeutic, preventive or diagnosticpurpose. Thus, the term “biologically active protein” comprises anyprotein or polypeptide, or fragment of a protein or polypeptide thatdisplays a useful biological activity in a mammal to which it isadministered, and in general the term protein as used herein embracespolypeptides and fragments of proteins and polypeptides. Thebiologically active protein moiety may be derived from a protein whichis endogenous to the mammal to which the molecule is administered, butmay just as well be a heterologous protein or an engineered protein. Thebiologically active protein moiety may be a soluble molecule or may bindto a receptor. Non-limiting examples of such activities are discussedbelow.

The activity of the biologically active protein may reside in an abilityto interact with a certain target molecule in the body of the mammal inquestion. Suitably, this target molecule is not a serum albumin of themammal. There are several examples of biologically active proteins thatrecognize and bind to target molecules, such as receptors and otherproteins, that are present on the surface of cells, or that recognizeand bind to target molecules within any of the cells variouscompartments, or that recognize and bind to target molecules that arepresent in extracellular body fluids.

Such biologically active proteins have, for example, been shown to beuseful in binding to target molecules that are preferentially present onthe surface of tumor cells or cancerous cells. A multitude of cancertargets or tumor targets have been described, as has a plethora ofantibodies, antibody fragments and other binding molecules with affinityfor these targets. As examples of such targets, mention is made of HER2(involved in certain forms of breast cancer), of CD4, CD20, CD22 andCD74 (all involved in different varieties of lymphoma), and of CEA andEpCAM (present on certain forms of solid tumors). A biologically activeprotein in the context of the present invention may for example be aprotein with an ability to interact with HER2, CD4, CD₂₀, CD22, CD74,CEA or EpCAM as a target molecule.

Further target molecules include toxins. For example a snake venom toxinmay be a suitable target and methods of the invention employed todeliver a biologically active moiety which neutralizes the toxin withoutstimulating a further immune response.

The biologically active protein moiety of the molecule used inaccordance with the present invention may also interact with moleculesthat are not cell-bound, and/or not involved in cancer. Thus, thebiologically active protein may for example be a protein that has theability to block an enzyme, for example to block enzymes involved in theblood clotting cascade or to block elastase. The biologically activeprotein may, alternatively, have the ability to block hormone orcytokine receptors.

As outlined immediately above, the biologically active protein may beselected from groups of proteins having the ability to interact with agiven target molecule. A non-limiting list of such proteins comprisesantibodies and fragments and derivatives thereof, staphylococcal proteinA and fragments and derivatives thereof, fibronectin and fragments andderivatives thereof, lipocalin and fragments and derivatives thereof,transferrin and fragments and derivatives thereof, and lectins andfragments and derivatives thereof. The naturally occurring forms of manyof these molecules have been subjected to protein engineeringtechniques, such as mutations and alterations in site-directed orrandomized approaches, with a view to create novel properties, such asbinding affinities for target molecules to which the naturally occurringform does not bind. Any such variant, or derivative, of the proteinslisted above may naturally be used as the biologically active proteinmoiety in the method or use according to the invention. A fragment ofany such molecule, whether of the naturally occurring form or of anengineered variant thereof, is also encompassed by the definition,insofar as the activity of the full-length protein is substantiallyretained in the fragment.

Further suitable biologically active proteins include growth hormone(GH), especially human growth hormone, ciliary neurotrophic factor(CNTF), granulocyte-macrophage colony stimulating factor (GM-CSF),insulin, interferon beta (IFN-β), factor VIII, erythropoietin, GL1P, andthrombopoietin.

The biologically active protein may therefore be staphylococcal proteinA, or a fragment or derivative thereof. For example, the IgG-binding Bdomain, or the Z protein derived therefrom (see Nilsson et al (1987),Prot Eng 1, 107-133, and U.S. Pat. No. 5,143,844), may be useful as thebiologically active protein moiety. Based on the Z protein as a basicstructure or scaffold, variants with an altered binding affinity havebeen selected from a library created by random mutagenesis in acombinatorial approach. Such proteins have been characterized in severalreports, and commercialized under the designation AFFIBODY® molecules.Representative publications include U.S. Pat. No. 6,534,628, Nord K etal, Prot Eng 8:601-608 (1995) and Nord K et al, Nat Biotech 15:772-777(1997). Such variants of protein Z derived from staphylococcal protein Aare useful as the biologically active protein moiety within the contextof the present invention.

The biologically active protein may alternatively, or additionally,exhibit a useful biological activity other than binding to a certaintarget molecule. For example, it may exhibit an enzymatic activity or ahormone activity.

All of the activities exemplified above may of course have atherapeutic, preventive or diagnostic effect. Thus, it is evident thatthe activity of the biologically active protein in some cases may bedescribed as a pharmaceutical activity.

Above, the at least two moieties of the molecule to be administered incarrying out the present invention are described as deriving from twodifferent molecular species, which have been coupled covalently in orderto provide the molecule which exhibits a reduced or non-existent immuneresponse upon administration. However, it is also contemplated that thetwo functions of i) biologically useful activity and ii) albumin bindingability may be combined within one and the same molecular species. Theuse as claimed herein of such molecules also falls within the scope ofthe present invention. An example of a molecule that illustrates thissituation is a protein with a first site, at which site the biologicallyuseful activity resides, and a second site, which is an albumin bindingsite. In other words the molecule consists of a protein with a firstsite, which site has a biologically useful activity and corresponds tothe biologically active protein, and a second site, which mediates thecapability of binding to a serum albumin of a mammal. The biologicallyuseful activity could then be any of the activities discussed above inrelation to the biologically active protein moiety. The two sites arespatially separated, and could for example reside on different faces ofa protein. A specific such protein is constituted by an albumin-bindingdomain from streptococcal protein G, which has been provided with anadditional binding site at another location on the molecule's surface.At this additional site, the albumin-binding domain may for example havebeen provided with an ability to interact with any one of the targetsdiscussed above. This additional binding site could be provided throughthe site-directed or random introduction of amino acid mutations in theprotein, such as addition, deletion or replacement of amino acidresidues. The resulting protein will exhibit a reduced or eliminatedimmunogenicity, as compared to the immunogenicity of a protein which issimilar in all respects except for the fact that the albumin-bindingfunction is not present.

According to a further aspect of the invention there is provided theconstructs Z_(Taq4:1)-ABD, ABD-Z_(her2:4), ABD-(Z_(her2:4))₂,ABD-(Z_(her2:4))₃, ABD-(Z_(her2:4))₄, (Z_(Aβ3))₂, ABD- and (Z_(Aβ3))₂.

According to a further aspect of the invention there is provided acomposition comprising a molecule comprising at least one moiety whichis a biologically active protein and at least one moiety capable ofbinding to a serum albumin of a mammal, for the preparation of amedicament which elicits no or a reduced immune response uponadministration to the mammal, as compared to the immune responseelicited upon administration to the mammal of the biologically activeprotein per se, as described above and a target molecule or portion oranalogue thereof which the biologically active protein binds to orotherwise interacts with. Preferably the target molecule is bound to thebiologically active protein moiety.

The invention will now be exemplified by the disclosure of experimentscarried out in accordance therewith and with reference to theaccompanying drawings FIGS. 1 to 25. The examples are not to beinterpreted as limiting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows ELISA titration curves for plasma from mice injected withHis₆-Z_(Taq4:1) as described in Example 1, when analyzed on ELISA platescoated with His₆-Z_(Taq4:1).

FIG. 2 shows ELISA titration curves for plasma from mice injected withZ_(Taq4:1)-ABD as described in Example 1, when analyzed on ELISA platescoated with Z_(Taq4:1)-ABD. In the panel showing plasma titres at Day 34of the experiment, a standard curve has been included.

FIG. 3 shows ELISA titration curves for IgG purified from plasma frommice injected with Z_(Taq4:1)-ABD as described in Example 1, whenanalyzed on ELISA plates coated with ABD or His₆-Z_(Taq4:1).

FIG. 4 shows ELISA titration curves for plasma from mice injected withstreptokinase as described in Example 1, when analyzed on ELISA platescoated with streptokinase.

FIG. 5 shows ELISA titration curves for plasma from mice injected withHis₆-Z_(Taq4:5) following scheme 1 as described in Example 2, whenanalyzed on ELISA plates coated with HiS₆-Z_(Taq4:5).

FIG. 6 shows ELISA titration curves for plasma from mice injected withHis₆-Z_(Taq4:5) following scheme 2 as described in Example 2, whenanalyzed on ELISA plates coated with HiS₆-Z_(Taq4:5).

FIG. 7 shows ELISA titration curves for plasma from mice injected withZ_(Taq4:1)-ABD following scheme 1 as described in Example 2, whenanalyzed on ELISA plates coated with Z_(Taq4:1)-ABD.

FIG. 8 shows ELISA titration curves for plasma from mice injected withZ_(Taq4:1)-ABD following scheme 2 as described in Example 2, whenanalyzed on ELISA plates coated with Z_(Taq4:1)-ABD.

FIG. 9 shows ELISA titration curves for plasma from mice injected withABD following scheme 1 (panel A) or scheme 2 (panel B) as described inExample 2, when analyzed on ELISA plates coated with ABD.

FIG. 10 shows ELISA titration curves for plasma from mice injected withHis₆-Z_(Taq4:5) as described in Example 3, when analyzed on ELISA platescoated with His₆-Z_(Taq4:5).

FIG. 11 shows ELISA titration curves for plasma from mice injected withABDZ_(her2:4) as described in Example 3, when analyzed on ELISA platescoated with ABD-(Z_(her)2:4)₂. The peaks seen in the diagrams of plasmafrom Day 7 and Day 14 are due to problems with the ELISA plate washer.

FIG. 12 shows ELISA titration curves for plasma from mice injected withABD-(Z_(her)2:4)₂ as described in Example 3, when analyzed on ELISAplates coated with ABD-(Z_(her)2:4)₂. The peak seen in the diagram ofplasma from Day 7 are due to problems with the ELISA plate washer.

FIG. 13 shows ELISA titration curves for plasma from mice injected withABD-(Z_(her2:4))₃ as described in Example 3, when analyzed on ELISAplates coated with ABD-(Z_(her)2:4)₂. The peak seen in the diagram ofplasma from Day 7 are due to problems with the ELISA plate washer.

FIG. 14 shows ELISA titration curves for plasma from mice injected withABD-(Z_(her)2:4)₄ as described in Example 3, when analyzed on ELISAplates coated with ABD-(Z_(her2:4))₂. The peaks seen in the diagram ofplasma from Day 7 are due to problems with the ELISA plate washer.

FIG. 15 shows ELISA titration curves for plasma from mice injected with(Z_(aβ:3))₂ as described in Example 4 when analyzed on ELISA platescoated with (Z_(aβ:3))₂.

FIG. 16 shows ELISA titration curves for plasma from mice injected withABD-(Z_(aβ:3))₂ as described in Example 4 when analyzed on ELISA platescoated with ABD-(Z_(aβ:3))₂.

FIG. 17 shows the results of Inhibition ELISA experiments described inExample 4.

FIG. 18 shows ELISA titration curves for plasma from mice injected withHis₆-Z_(Taq4:1) as described in Example 5 when analyzed on ELISA platescoated with His₆-Z_(Taq4:1).

FIG. 19 shows ELISA titration curves for plasma from mice injected withZ_(Taq4:1)-ABD as described in Example 5 when analyzed on ELISA platescoated with Z_(Taq4:1)-ABD.

FIG. 20 shows ELISA titration curves for plasma from mice injected withHis₆-Z_(Taq4:1) as described in Example 5 when analyzed on ELISA platescoated with His₆-Z_(Taq4:1).

FIG. 21 shows ELISA titration curves for plasma from mice injected withZ_(Taq4:1) ABD and then His₆-Z_(Taq4:1) as described in Example 5.

FIG. 22 shows ELISA titration curves for plasma from mice injected withZ_(Taq4:1)-ABD and then Z_(Taq4:1)-ABD and His₆-Z_(Taq4:1) as describedin Example 5.

FIG. 23 shows ELISA titration curves for plasma from mice injected withZ_(Taq4:1) and then Z_(Taq4:1)-ABD as described in Example 5.

FIG. 24 shows ELISA titration curves for plasma from rats injected with(ZAβ3)₂ as described in Example 6.

FIG. 25 shows ELISA titration curves for plasma from rats injected withABD(ZAβ3)₂ as described in Example 6.

FIG. 26 is a table giving the sequence of various constructs inaccordance with the invention.

FIG. 27 A is a summary of the set up of Example 5; FIG. 27B illustratesresults obtained in Example 5.

EXAMPLE 1 Humoral Immune Response in Mice Following Administration ofVarious Molecules Molecules Studied

In this Example, the inventive concept was studied through a comparisonof the antibody response in mice upon administration of differentmolecules. The molecules administered were the following:

His₆-Z_(Taq4:1)—a variant of protein Z, in turn derived from the Bdomain of staphylococcal protein A. This Z variant was produced usingrecombinant DNA technology, through the expression of the DNA sequenceencoding it, and simultaneously provided with a hexahistidyl tagaccording to known molecular biology procedures. Z variant Z_(Taq4:1)has previously been selected on the basis of its affinity for Taq DNApolymerase. A description of the Z_(Taq4:1) molecule, including itsamino acid sequence and the procedure for selection thereof, is given inGunneriusson E et al, Protein Eng 12: 10, 873-878 (1999) (see e.g. FIG.1 in this article). Used for comparative purposes.

Z_(Taq4:1)-ABD—a fusion protein between the Z variant Z_(Taq4:1) and the46 amino acid albumin binding domain (ABD) of streptococcal protein Gstrain G148 (Kraulis P J et al, FEBS Lett 378:190 (1996))). The fusionprotein was prepared through expression of the corresponding DNAsequence in accordance with known molecular biology procedures. Used toillustrate the invention.

Streptokinase—a commercially available bacterial enzyme known to inducestrong antibody responses. Purchased from Sigma (cat no S-8026, lot092K₁₅₁₅) and used as a positive control.

Materials and Methods

Mice and Administration Schedule:

Female NMRI mice (20 mice, plus 2 as a reserve) were used in thisexperiment. Body weight upon arrival was 20 g. At the start of theimmunization experiments, the mice were from 8 to 12 weeks old. The micewere kept and fed in accordance with guidelines from the SwedishMinistry of Agriculture, Food and Fisheries. Food and water were givenad libitum. For the immunization experiment, the mice were split intofive groups with four animals in each group. 20 μg of the moleculesindicated in Table 1 were administered subcutaneously to each mouse in0.1 ml NaCl, 0.9%.

TABLE 1 Mouse groups and administered molecules Group Mouse # Molecule 11-4 His₆-Z_(Taq4:1) 2 5-8 Z_(Taq4:1)-ABD 3 17-20 Streptokinase

The solutions of test molecules were kept frozen at −20° C. and thawedbefore injection. Repeated injections were given at Day 0, 3, 6, 9, 12and 21 of the study. Blood samples of 150 μl were taken from the orbitalplexus of the mice at Day 0 (preplasma), 7, 14 and 21 of the study. AtDay 34 of the study, the mice were sacrificed and the maximal amount ofblood obtained. Blood was collected in K⁺EDTA tubes, and left to standfor one hour after sampling. Thereafter, samples were centrifuged at6000 rpm for 6 min in order to separate plasma. Plasma was frozen at−20° C. for storage before analysis.

Analysis of Plasma Samples by Specific ELISA:

For analysis of plasma from mice receiving one of the molecules above,ELISA-plates (Costar, no 9018) were coated with the correspondingmolecule diluted in coating buffer (15 mM Na₂HCO₃, 35 mM NaHCO₃, pH 9.6)to a final concentration of 1 μg/ml. 100 μl of the coating solution wasadded per well and plates were incubated for 1-3 nights at 4° C. Theplates were then washed manually 3 times with deionized water andblocked with 200 μl/well, using PBS (2.68 mM KCl, 1.47 mM KH₂PO₄, 137 mMNaCl, 8.1 mM Na₂HPO₄, pH 7.4) with either 1% bovine serum albumin (BSA;from Sigma, cat no A-2153) or 2% dry milk (Semper AB, Stockholm,Sweden), for 1 to 2 hours. The blocking solution was then removed and100 μl plasma was added to each well, diluted from 1:100 in blockingsolution and then in a series of 2-fold dilutions. After 2 hours ofincubation, plates were washed manually 3 times with PBS-T (PBS with0.05% Tween 20; Tween 20 from Acros Organics, cat no 233362500).Thereafter, 100 μl of a secondary antibody, HRP-conjugated goatanti-mouse IgG (Southern Biotech no 1031-05), diluted 1:2000 in blockingsolution, was added to each well. Plates were incubated for 1 hour. Thethree steps (blocking, addition of plasma sample, addition of secondaryantibody) were performed on a shaker, and the last step in the dark. Theplates were washed manually 5 times with PBS-T. Subsequently, 100 μl ofa substrate solution (ImmunoPure® TMB; Pierce, cat no 34021) was addedto each well and plates were subsequently incubated in the dark. Colordevelopment was stopped after 15 minutes by addition of 100 n1 stopsolution (2 M H₂SO₄; VWR, cat no 14374-1). Plates were read at 450 nm inan ELISA spectrophotometer (Basic Sunrise, Tecan).

Also included was a standard comprising a pool of mouse plasmapreviously obtained, containing 210 μg/ml of anti-His₆-Z_(Taq4:1) IgG.This pool was used in 2-fold dilution series. The limit of detection inELISA plates coated with His₆-Z_(Taq4:1) was approximately 5 ng/ml.

Analysis of Total IgG Content in Plasma Samples:

For determination of the total amount of IgG in the samples,quantitative ELISA analyses were performed using a Mouse IgG ELISAQuantitation kit (Bethyl, cat no E90-131), according to themanufacturer's instructions. In brief, ELISA plates were coated withanti-mouse IgG provided by the manufacturer. The plasma to beinvestigated and the standard plasma were added in 2-fold dilutionseries beginning with 2000 ng/ml. The detection antibody was diluted1:100000 (anti-mouse IgG, HRP conjugate; provided by the manufacturer)in blocking buffer. The ELISA analyses were developed using ImmunoPure®TMB as described above.

Purification of IgG:

IgG was purified from a pool of Day 34 plasma obtained from miceinjected with Z_(Taq4:1)-ABD. The pool was diluted 12.5 times with PBS-Tbefore loading on a HiTrap column provided with an IgG-specific affinityligand derived from staphylococcal protein A. The column had beenpreviously equilibrated with PBS-T. The column was washed untilabsorbance values reached zero, and bound IgG was eluted using elutionbuffer (0.2 M glycine, 1 mM EGTA, pH 2.8). For neutralization, Tris baseto a concentration of 50 mM and 1/10 volume of 10×PBS were added.

Data Analysis:

ELISA values were obtained using the Magellan software from Tecan. Thevalues were exported to Microsoft Excel for analysis. IgG concentrationswere calculated by comparison with a standard curve using the Xlfit.3program (www.idbs.com). The values/curves obtained with the standardpool of anti-His₆-Z_(Taq4:1) IgG and the Bethyl standard plasma wereused for determination of specific and total IgG, respectively.

Results

His₆-Z_(Taq4:1):

Mice #1-#4 were injected with His₆-Z_(Taq4:1). Plasma samples obtainedbefore immunization (preplasma), and after 7, 14, 21 and 34 days wereanalyzed for presence of His₆-Z_(Taq4:1) specific antibodies, asdescribed in the method section. ELISA plates were blocked with 1% BSA.There were no antibodies against the injected molecule in preplasma. Allfour mice responded by generating a moderate antibody response againstthe injected protein. The titre of specific antibodies rose steadilyduring the treatment, with the highest titre at Day 34. FIG. 1 shows thetitration curves of individual plasma samples, plotted against OD valuesat 450 nm.

Total IgG concentration was determined using the Bethyl-ELISA, and theconcentration of IgG specific for His₆-Z_(Taq4:1) was determined usingthe standard pool as described above. The results are presented in Table2. The concentration of specific antibodies increased 50-500 timesduring the 34 days of treatment. Taking the total IgG concentration intoaccount, the specific antibodies constituted about 1-4% of the totalamount of IgG.

TABLE 2 Total and specific IgG in plasma following administration ofHis₆-Z_(Taq4:1) Total IgG (μg/ml) Specific IgG (μg/ml) Mouse Mol- Day: #ecule 0 34 0 7 14 21 34 1 His₆- 2100 550 ND* ND* ND* 3.4 5.2 Z_(Taq4:1)2 His₆- 320 1200 ND* ND* 13 21 29 Z_(Taq4:1) 3 His₆- 350 900 ND* ND* 1.813 37 Z_(Taq4:1) 4 His₆- 810 740 ND* ND* 6.9 12 17 Z_(Taq4:1) *ND—Notdetectable (below detection limit)

Z_(Taq4:1)-ABD:

Mice #5-#8 were injected with Z_(Taq4:1)-ABD. Plasma samples obtainedbefore immunization (preplasma, data not shown), and after 7, 14, 21 and34 days were analyzed for presence of antibodies specific forZ_(Taq4:1)-ABD, as described in the method section. ELISA plates wereblocked with 1% BSA. FIG. 2 shows the results obtained from individualplasma samples, plotted against OD values at 450 nm. In summary, nospecific antibodies were detected against the injected Z_(Taq4:1)-ABD.

The risk that mouse serum albumin interacted with the ABD moiety of theprotein coated in the ELISA wells, and thus sterically hindered mouseantibodies from specific binding to the Z_(Taq4:1)-ABD molecules, wastested. In the first approach, ABD was avoided by coating withHis₆-Z_(Taq4:1). Plasma from day 34 were tested, and results showed nospecific interaction between mouse antibodies and His₆-Z_(Taq4:1)(datanot shown). Another approach was to remove albumin from the plasma.Total IgG was therefore purified from a pool of Day 34 plasma from mice#5-#8 using a HiTrap column as described in the “Materials and methods”section. The IgG fraction was tested on plates coated with ABD orHis₆-Z_(Taq4:1), and the results are shown in FIG. 3. In summary, nobinding could be detected between mouse antibodies and theHis₆-Z_(Taq4:1) or ABD coated surfaces.

Total IgG concentration was determined using the Bethyl-ELISA asdescribed above, and the results are presented in Table 3.

TABLE 3 Total IgG in plasma following administration of Z_(Taq4:1)-ABDTotal IgG (μg/ml) Day: Mouse # Molecule 0 34 5 Z_(Taq4:1)-ABD 270 320 6Z_(Taq4:1)-ABD 280 370 7 Z_(Taq4:1)-ABD 1000 950 8 Z_(Taq4:1)-ABD 14001200

Streptokinase:

Mice #17-#20 were injected with streptokinase, a bacterial enzyme thatprevents clotting of blood and is known to induce strong antibodyresponses. Plasma samples obtained before injection (preplasma, notshown), and after 7, 14, 21 and 34 days were analyzed for the presenceof streptokinase-specific antibodies, as described in themethod-section. The ELISA plates were blocked with 2% dry milk. FIG. 4shows the results obtained from individual plasma samples, plottedagainst OD values at 450 nm. As shown in FIG. 4, streptokinase inducedan antibody response that increased during the treatment period. At Day34, high titres of streptokinase-specific antibodies were detected inall four mice.

Total IgG concentration was determined using the Bethyl-ELISA asdescribed above, and the results are presented in Table 4. The total IgGconcentration was in all but one case considerably higher at deathbleeding than in preplasma, implying a normal maturation of the immunesystem and/or an ongoing immune response.

TABLE 4 Total IgG in plasma following administration of streptokinaseTotal IgG (μg/ml) Day: Mouse # Molecule 0 34 17 Streptokinase 1500 110018 Streptokinase 1200 4800 19 Streptokinase 3000 6300 20 Streptokinase280 4100

Endpoint Titres:

The concentration of antibodies specific for the administered moleculewas determined only in plasma from mice injected with His₆-Z_(Taq4:1),due to the lack of suitable standards for the other proteins. For thisreason, endpoint titres were used for comparisons between differentinjected proteins. The endpoint titre was defined as the dilution wherethe OD value equaled two times the background, the background being theOD value obtained in preplasma from the same mouse. Table 5 shows theendpoint titres for each mouse. To summarize the results, the endpointtitres after immunization with streptokinase were considerably higherthan those obtained with different Z variants. Of particular relevanceto the present invention was that antibody titres after immunizationwith Z_(Taq4:1) were not detectable.

TABLE 5 Endpoint titres of antibodies following administration ofvarious molecules to mice His₆-Z_(Taq4:1) Z_(Taq4:1)-ABD StreptokinaseEndpoint Endpoint Endpoint Mouse # titre Mouse # titre Mouse # titre 12700 5 No value 17  78 000 2 14 000 6 No value 18 Value out of range 316 000 7 No value 19 110 000 4 8000 8 No value 20 730 000

Discussion

To study the effect of administration of different molecules oninitiation of specific B-cell activation and antibody generation in ananimal model, NMRI mice were injected subcutaneously with differentmolecules. In order to simulate a normal treatment cycle, six injectionsof adequate doses without adjuvant were administered to the mice (threedays apart, with a booster injection at Day 21). Plasma from treatedanimals were analyzed for the presence of antibodies of IgG isotype withspecificity for the injected molecules. In addition, levels of total IgGcontent of all animals were determined in preplasma and on the day ofdeath bleeding (Day 34).

As shown in the “Results” section above, His₆-Z_(Taq4:1) inducedspecific IgG responses in the studied animals. The antibody responseswere low to moderate (5 to 37 μg/ml at Day 34), with the most apparentincrease after booster injection at Day 21 (2-20 times increase). Theresponses peaked at Day 34.

Importantly, plasma from animals injected with Z_(Taq4:1)-ABD did notshow any specific binding to the injected molecules as determined byELISA. This result was in great contrast to the specific IgG titresobserved when analyzing His₆-Z_(Taq4:1), i e the same Z sequence fusedto His₆ instead of ABD. All five time-points analyzed were negative fortarget-specific mouse IgG antibodies. The Limit of Detection (LOD) inthe ELISA was about 5 ng/ml.

Streptokinase, a bacterial protein considered to be a strong immunogen,was used as positive control in the studies. As expected, high specificIgG responses were observed in animals injected with streptokinase.

In conclusion, the injected proteins can be ranked in the followingmanner, according to their ability to evoke specific IgG responses:streptokinase>>His₆-Z_(Taq4:1)>>Z_(Taq4:1)-ABD. The most interesting andrelevant result was that the fusion of ABD to Z_(Taq4:1) resulted in aspecific IgG response that could not be detected.

EXAMPLE 2 Humoral Immune Response in Mice Following Administration ofDifferent Molecules at Various Frequencies Molecules Studied

In this Example, the inventive concept was again studied through acomparison of the antibody response in mice upon administration ofdifferent molecules. The molecules administered were the following:

His₆-Z_(Taq4:5)—a variant of protein Z, in turn derived from the Bdomain of staphylococcal protein A. The Z_(Taq4:5) variant was producedusing recombinant DNA technology, through expression of the DNA sequenceencoding it, and simultaneously provided with a hexahistidyl tagaccording to known molecular biology procedures. Z_(Taq4:5), ineludingits selection and amino acid sequence, is described in Gunneriusson E etal, supra, where it is denoted Z_(Taq S1-1). Used for comparativepurposes.

Z_(Taq4:1)-ABD—as described in Example 1. Used to illustrate theinvention.

ABD—the albumin binding domain (ABD) of streptococcal protein G strainG148 (see above for references). Prepared through expression of thecorresponding DNA sequence in accordance with known molecular biologyprocedures. Used for comparative purposes.

Materials and Methods

Mice and Administration Schedule:

Female NMRI mice (40 mice, plus 2 as a reserve) were used in thisexperiment. Body weight upon arrival was 20 g. At the start of theimmunization experiments, the mice were from 8 to 12 weeks old. The micewere kept and fed in accordance with guidelines from the SwedishMinistry of Agriculture, Food and Fisheries. Food and water were givenad libitum. For the immunization experiment, the mice were split intoseven groups according to Table 6. 20 μg of the molecules indicated inTable 6 were administered subcutaneously to each mouse in 0.1 ml NaCl,0.9%.

TABLE 6 Mouse groups and administered molecules Group Mouse # Molecule 153-60 His₆-Z_(Taq4:5) 2 61-68 Z_(Taq4:1)-ABD 3 69-76 ABD 4 82-85His₆-Z_(Taq4:5) 5 86-89 Z_(Taq4:1)-ABD 6 90-93 ABD

The solutions of test molecules were kept frozen at −20° C. and thawedbefore injection. The mice of groups 1-3 received subcutaneousinjections at Day 0, 7 and 21 of the study (scheme 1, low frequency).The mice of groups 4-6 received subcutaneous injections at Day 0, 1 and21 of the study (scheme 2, high frequency). Blood samples of 150 μl weretaken from the orbital plexus of the mice at Day 0 (preplasma), 7, 14and 21 of the study. At Day 34 of the study, the mice were sacrificedand the maximal amount of blood obtained. Blood was collected in K⁺EDTAtubes, and left to stand for one hour after sampling. Thereafter,samples were centrifuged at 6000 rpm for 6 min in order to separateplasma. Plasma was frozen at −20° C. for storage before analysis.

Analysis of Plasma Samples by Specific ELISA:

For analysis of plasma from mice receiving one of the molecules above,ELISA-plates (Costar, no 9018) were coated with the correspondingmolecule diluted in coating buffer (15 mM Na₂HCO₃, 35 mM NaHCO₃, pH 9.6)to a final concentration of 1 μg/ml. 100 μl of the coating solution wasadded per well and plates were incubated for 1-3 nights at 4° C. Theplates were then washed manually 3 times with deionized water andblocked with blocking buffer (200 μl/well; PBS (2.68 mM KCl, 1.47 mMKH₂PO₄, 137 mM NaCl, 8.1 mM Na₂HPO₄, pH 7.4) with 0.5% casein (Sigma,cat no C-8654), for 1 to 2 hours. The blocking buffer was then removedand 100 μl plasma was added to each well, diluted from 1:100 in blockingsolution and then in a series of 2-fold dilutions. Also included was astandard comprising a pool of mouse plasma previously obtained,containing 210 μg/ml of antiHis₆-Z_(Taq4:1) IgG. This pool was used in2-fold dilution series. The limit of detection in ELISA plates coatedwith His₆-Z_(Taq4:1) was approximately 5 ng/ml.

After 2 hours of incubation, plates were washed manually 3 times withPBS-T (PBS with 0.05% Tween 20). Thereafter, 100 μl of a secondaryantibody, HRP-conjugated goat anti-mouse IgG (Southern Biotech no1031-05), diluted 1:2000 in blocking buffer, was added to each well.Plates were incubated for 1 hour. All steps were performed on a shaker,and the last step in the dark. The plates were washed manually fivetimes with PBS-T. Subsequently, 100 μl of a substrate solution(ImmunoPure® TMB; Pierce, cat no 34021) was added to each well andplates were subsequently incubated in the dark.

Color development was stopped after 15 minutes by addition of 100 n1stop solution (2 M H₂SO₄; VWR, cat no 14374-1). Plates were read at 450nm in an ELISA spectrophotometer (Basic Sunrise, Tecan) using Magellansoftware.

Analysis of Total IgG Content in Plasma Samples:

For determination of the total amount of IgG in the samples,quantitative ELISA analyses were performed using a Mouse IgG ELISAQuantitation kit (Bethyl, cat no E90-131), according to themanufacturer's instructions. In brief, ELISA plates were coated withanti-mouse IgG (1 μg/ml) provided by the manufacturer. The plasma to beinvestigated and the standard plasma were added in 2-fold dilutionseries. The standard plasma was diluted from 2000 ng/ml. The detectionantibody was used at a dilution of 1:100000 (anti-mouse IgG, HRPconjugate; provided by the manufacturer) in blocking buffer. The ELISAanalyses were developed using ImmunoPure® TMB as described above.

Data Analysis:

ELISA values were obtained using the Magellan software from Tecan.

The values were exported to Microsoft Excel for analysis. IgGconcentrations were calculated by comparison with a standard curve usingthe Xlfit.3 program (www.idbs.com). The values/curves obtained with thestandard pool of anti-His₆-Z_(Taq4:1) IgG and the Bethyl standard plasmawere used for determination of specific and total IgG, respectively.

Results

His₆-Z_(Taq4:5):

Eight mice (#53-#60) were injected with His₆-Z_(Taq4:5) at 20 mg/mouseand injection, according to scheme 1 (see above). Plasma samplesobtained before (preplasma), during (Day 7, 14, 21) and after (Day 34)injections were analyzed for presence of His₆-Z_(Taq4:5) specificantibodies as described above. The results are shown in FIG. 5. Therewere no His₆-Z_(Taq4:5) specific antibodies in preplasma (data notshown) or at Day 7 (FIG. 5). The number of positive plasma had increasedby Day 14 and 21. At Day 34, all plasma samples containedHis₆-Z_(Taq4:5) specific antibodies, although the level variedconsiderably between individual mice.

Four mice (#82-#85) were injected with His₆-Z_(Taq4:5) according toscheme 2 (see above).

Plasma samples obtained before (preplasma), during (Day 7, 14, 21) andafter (Day 34) injection were analyzed for presence of His₆-Z_(Taq4:5)specific antibodies as described above. The results are shown in FIG. 6.There were no His₆-Z_(Taq4:5) specific antibodies in preplasma (data notshown). Antibodies against HiS₆-Z_(Taq4:5) were found at low levels insome, but not all, plasma samples already from Day 7. The number ofpositive plasma samples and the titre did not increase at Day 14 and 21as for scheme 1. At Day 34, all but one plasma sample had levels ofHis₆-Z_(Taq4:5) specific antibodies that were high compared to those ofDay 21.

The concentration of specific IgG was determined using the standard poolof anti-His₆-Z_(Taq4:1)IgG (see method section). This pool haspreviously been shown to contain 210 μg/ml of His₆-Z_(Taq4:1) specificantibodies. Concentrations were calculated using the XLfit program andthe one-site, dose-response formula. Both samples and standard weretested as singles, and the standard variation of the method is thereforeunknown. In addition, the mathematical formula chosen for calculationalso affects the values, and the variation depending on the formula hasnot been calculated. Thus, the concentrations given in the table belowshould be considered as relative, rather than absolute, values. Table 7shows the concentration of His₆-Z_(Taq4:5) specific antibodies in plasmafrom individual mice. As shown in the titration analysis above, theconcentration of specific IgG at Day 34 varied considerably betweenindividual mice in both groups.

The seemingly higher concentration in the scheme 1 group was notstatistically significant when tested with Student's T test (TTESTfunction, Microsoft Excel).

Total IgG concentrations were also determined in Day 34 plasma samplesfrom the His₆-Z_(Taq4:5) injected mice, using quantitative ELISA asdescribed above. The results are presented in Table 7 (column 7).

TABLE 7 Total and specific IgG in plasma following administration ofHis₆-Z_(Taq4:5) Total % specific Specific IgG (μg/ml) IgG IgG of totalMouse Day Day Day Day (μg/ml) IgG Scheme # 7 14 21 34 Day 34 Day 34 1 53 ND* 20 20 260 1100 26 54 ND ND ND 20 1500 1 55 ND ND 20 160 1500 9 56ND 30 30 90 740 9 57 ND 50 50 10 1500 1 58 ND ND ND 1800 2000 80 59 NDND ND 6 680 1 60 ND ND ND 60 510 12 2 82 ND ND ND 100 3430 3 83 ND ND NDND 3430 0.01 84 ND ND ND 10 2483 0.4 85 ND ND ND 20 6400 0.3 *ND—Notdetectable (below detection limit)

Z_(Taq4:1)-ABD:

Eight mice (#61-#68) were injected with Z_(Taq4:1)-ABD using scheme 1.Plasma samples obtained before injection (preplasma, data not shown) andafter 7, 14, 21 and 34 days were analyzed for presence of Z_(Taq4:1)-ABDspecific antibodies as described above. FIG. 7 shows the results. As isevident from FIG. 7, no specific IgG were induced againstZ_(Taq4:1)-ABD.

There are high levels of mouse serum albumin (MSA) present in theanalyzed samples. To circumvent the problem with MSA that might bind tothe Z_(Taq4:1)-ABD coated ELISA surface, plasma samples were alsotitrated on plates coated with His₆-Z_(Taq4:1) (data not shown). Theanalyses were once again negative, confirming the observation that theZ_(Taq4:1)-ABD molecule is non-immunogenic.

Four mice (#86-#89) were injected with Z_(Taq4:1)-ABD according toscheme 2. Plasma samples obtained before injection (preplasma, data notshown) and after 7, 14, 21 and 34 days were analyzed for presence ofZ_(Taq4:1)-ABD specific antibodies as described above. The results areshown in FIG. 8. Again, no IgG response could be measured.

Total IgG concentration was determined using the Bethyl-ELISA asdescribed above, and the results are presented in Table 8.

TABLE 8 Total IgG in plasma following administration of Z_(Taq4:1)-ABDScheme Mouse # Total IgG (μg/ml) at Day 34 1 61  500 62 1300 63 2600 641800 65 1700 66 1100 67  830 68  380 2 86 2500 87  870 88 2900 89 11100**Unreliable, since value out of range

ABD:

Eight mice (#69-#76) were injected with ABD according to scheme 1.Plasma obtained before injection (preplasma) and after 7, 14, 21 and 34days was analyzed for presence of ABD-specific antibodies as describedabove. Very low titres of antibodies against the ABD molecule weredetected, and only in Day 34 plasma (FIG. 9A). Preplasma and plasma fromDay 7, 14 and 21 were negative (data not shown).

Four mice (#90-#93) were injected with ABD according to scheme 2. Plasmaobtained before injection (preplasma) and after 7, 14, 21 and 34 dayswas analyzed for presence of ABD-specific antibodies as described above.No antibody response could be measured (FIG. 9B shows the results forsamples of Day 34).

Total IgG concentration was determined using the Bethyl-ELISA asdescribed above, and the results are presented in Table 9.

TABLE 9 Total IgG in plasma following administration of ABD Scheme Mouse# Total IgG (μg/ml) at Day 34 1 69 750 70 400 71 940 72 410 73 390 74100 75 1100 76 304 2 90 3200 91 5100 92 6500 93 5800

Discussion

In accordance with the results of Example 1, Z_(Taq4:1)-ABD was unableto induce specific IgG responses in the mice. Likewise, theadministration of ABD itself, devoid of fusion partner, did not elicitany measurable specific IgG response. The animals in these groups wereadministered the same amount of protein as in Example 1. Neither lowfrequency or high frequency injections generated detectable IgGresponses. Since it is believed that the Z protein and its derivativesare T-dependent antigens, they would be expected to generate specificantibodies predominantly of the IgG isotype. Therefore, since theseresults show that no IgG antibodies are formed, they imply that noantibodies of other isotypes are formed either, in response to theZ_(Taq4:1)-ABD fusion protein.

EXAMPLE 3 Humoural Immune Response in Mice Following Administration ofMultimers of a Z Variant Provided with an Albumin Binding MoietyMolecules Studied

As in Examples 1 and 2, the inventive concept was again studied througha comparison of the antibody response in mice upon administration ofdifferent molecules. The molecules administered were the following:

His₆-Z_(Taq4:5)—as described in Example 2. Used for comparativepurposes.

ABD-Z_(her2:4)—a fusion protein between the albumin binding domain (ABD)of streptococcal protein G strain G148 (see above) and the Z variantZ_(her2:4). Z_(her2:4) was selected from a library of combinatorialvariants of Z and characterized. In the selection procedure, purifiedprotein corresponding to the cancer antigen HER2 (also described in theliterature as neu, HER2/neu or c-erbB-2) was used as the targetmolecule. Z_(her2:4) was found to interact with HER2 with a K_(D) valueof approximately 50 nM. The amino acid sequence of Z_(her2:4), instandard one-letter code, is:

(SEQ ID NO: 4) VDNKFNKELR QAYWEIQALP NLNWTQSRAF IRSLYDDPSQSANLLAEAKK LNDAQAPK

The fusion protein was prepared through expression of the correspondingDNA sequence together with the DNA sequence encoding the ABD moiety, inaccordance with known molecular biology procedures. The ABD-Z_(her2:4)fusion protein is used to illustrate the invention.

ABD-(Z_(her2:4))₂— a fusion protein between the albumin binding domain(ABD) and a dimer of the Z variant Z_(her2:4), prepared in accordancewith known molecular biology procedures with the added knowledge ofZ_(her2:4) sequence information. Used to illustrate the invention.

ABD-(Z_(her2:4))₃— a fusion protein between the albumin binding domain(ABD) and a trimer of the Z variant Z_(her2:4), prepared in accordancewith known molecular biology procedures with the added knowledge ofZ_(her2:4) sequence information. Used to illustrate the invention.

ABD-(Z_(her2:4))₄— a fusion protein between the albumin binding domain(ABD) and a tetramer of the Z variant Z_(her2:4), prepared in accordancewith known molecular biology procedures with the added knowledge ofZ_(her2:4) sequence information. Used to illustrate the invention.

Materials and Methods

Mice and Administration Schedule:

Female NMRI mice (30 mice, plus 2 as a reserve) were used in thisexperiment. Body weight upon arrival was 20 g. At the start of theimmunization experiments, the mice were from 8 to 12 weeks old. The micewere kept and fed in accordance with guidelines from the SwedishMinistry of Agriculture, Food and Fisheries. Food and water were givenad libitum. For the immunization experiment, the mice were split intofive groups according to Table 10. 20 μg of the molecules indicated inTable 10 were administered subcutaneously to each mouse in 0.1 ml NaCl,0.9%.

TABLE 10 Mouse groups and administered molecules Group Mouse # Molecule1 114-119 His₆-Z_(Taq4:5) 2 120-125 ABD-Z_(her2:4) 3 126-131ABD-(Z_(her2:4))₂ 4 132-137 ABD-(Z_(her2:4))₃ 5 138-143ABD-(Z_(her2:4))₄

The solutions of test molecules were kept frozen at −20° C. and thawedbefore injection. The mice of group 1 received subcutaneous injectionsat Day 0, 3, 6, 9, 12 and 63 of the study (scheme 1). Blood samples of150 μl were taken from the orbital plexus of the mice of group 1 at Day0 (preplasma), 7, 14, 21, 34, 49 and 63 of the study. At Day 73 of thestudy, these mice were sacrificed and the maximal amount of bloodobtained. The mice of groups 2-5 received subcutaneous injections at Day0, 3, 6, 9, 12 and 21 of the study (scheme 2). Blood samples of 150 μlwere taken from the orbital plexus of the mice of groups 2-5 at Day 0(preplasma), 7, 14 and 21 of the study. At Day 34 of the study, thesemice were sacrificed and the maximal amount of blood obtained. Blood wascollected in K⁺DTA tubes, and left to stand for one hour after sampling.Thereafter, samples were centrifuged at 6000 rpm for 6 min in order toseparate plasma. Plasma was frozen at −20° C. for storage beforeanalysis.

Analysis of Plasma Samples by Specific ELISA:

In general, a volume of 100 μl per well was used for all incubationsteps except for blocking, where 200 μl were used. ELISA plates (Costar,no 9018) were incubated 1-3 days for coating, 1-2 hours for blocking andplasma, 1 hour for secondary antibody and 15 min for substrate solution.The incubations were performed on a shaker at room temperature, exceptcoating, which was incubated at 4° C. Washing was done between allsteps, unless otherwise stated, using the ELISA SkanWasher 300 (Skatron)with 4×350 μl washing buffer (PBS-T, see Example 1) per well. Plateswere read at 450 nm in a Tecan ELISA reader using the Magellan v3.11software. Blocking buffer was used for all dilutions except coating,where coating buffer (15 mM Na₂HCO₃, 35 mM NaHCO₃, pH 9.6) was usedinstead.

ELISA-plates were coated with His₆-Z_(Taq4:5) or ABD-(Z_(her2:4))₂,diluted to a concentration of 5 μg/□ml. After coating, plates wereblocked with PBS+0.5% casein (PBS (2.68 mM KCl, 1.47 mM KH₂PO₄, 137 mMNaCl, 8.1 mM Na₂HPO₄, pH 7.4) with 0.5% casein (Sigma, cat no C-8654)).Blocking was removed and plasma was added, diluted from 1:100 and thenin 3-fold dilution series. Plasma from mice injected withHis₆-Z_(Taq4:5) was analyzed on ELISA plates coated withHis₆-Z_(Taq4:5), whereas plasma from mice injected with the fourdifferent Z_(her2:4) constructs was analyzed on ELISA plates coated withABD-(Z_(her2:4))₂. Also included was a standard comprising a pool ofmouse antibodies previously obtained. The pool included IgG directedagainst Z_(Taq4:5) and Z_(her2:4). HRP conjugated goat anti-mouse IgG(Southern Biotech, cat no 1031-05), diluted 1:2000, was used as thesecondary reagent, and the reaction was developed using ImmunoPure® TMBsubstrate solution (Pierce, cat no 34021). This incubation was performedin the dark. The colour development was stopped after 15 minutes by theaddition of stop solution (2 M H₂SO₄; VWR, cat no 14374-1).

Results

Plasma samples obtained before immunization (preplasma), and after 7,14, 21 and 34 days were analyzed for presence of specific antibodies, asdescribed in the “Materials and methods” section. There were noantibodies specific for the injected molecules in any preplasma (datanot shown). The diagrams presented in FIGS. 10-14 show the titrationcurves of individual plasma samples plotted against OD values at 450 nm.

His₆-Z_(Taq4:5):

The results are shown in FIG. 10. Mice injected with His₆-Z_(Taq4:5)showed high antibody responses. The response was time dependent andseemed to peak at Day 21. Previous experiments with His₆-Z_(Taq4:5)(Example 2) generally peaked at Day 34. The reason for the lower ODvalues at Day 34 of this study was likely due to the fact that theseanimals did not receive a booster injection at Day 21.

ABD-Z_(her2:4):

The results are shown in FIG. 11. Mice injected with ABD-Z_(her2:4)showed no specific IgG response. The peaks seen in the diagrams ofplasma from Day 7 and Day 14 are due to problems with the ELISA platewasher, and thus do not correctly represent the antibody content in theplasma.

ABD-(Z_(her2:4))₂:

The results are shown in FIG. 12. Mice injected with ABD-(Z_(her2:4))₂showed no specific IgG response. The peak seen in the diagram of plasmafrom Day 7 are due to problems with the ELISA plate washer, and thus donot correctly represent the antibody content in the plasma.

ABD-(Z_(her2:4))₃:

The results are shown in FIG. 13. Mice injected with ABD-(Z_(her2:4))₃showed no specific IgG response. The peak seen in the diagram of plasmafrom Day 7 are due to problems with the ELISA plate washer, and thus donot correctly represent the antibody content in the plasma.

ABD-(Z_(her2:4))₄:

The results are shown in FIG. 14. Mice injected with ABD-(Z_(her2:4))₄showed no specific IgG response. The peaks seen in the diagram of plasmafrom Day 7 are due to problems with the ELISA plate washer, and thus donot correctly represent the antibody content in the plasma.

Also tested was the ability of the four different ABD-(Z_(her2:4))_(n)constructs to elicit a specific IgM response. No such responses weredetected (data not shown).

Discussion

The results of this experiment confirm the finding from Examples 1 and 2that the provision of an albumin-binding moiety reduces or eliminatesthe immune response to a biologically active protein. Importantly, theeffect was shown to be valid for proteins of increasing size. Thetetramer of Z_(her2:4) comprises more than 230 amino acid residues, butdespite its size does not elicit a substantial antibody response uponadministration in mice.

EXAMPLE 4 Reduction of Immune Response Tested with Dimers of FurtherBiologically Active Molecules Molecules Studied

The aim of this study was also to assess if the antibodies, generatedafter immunization, are able to inhibit the binding between specificAFFIBODY® molecules and their target protein. The previous observationof a reduction in immune response when a biologically active protein iscoupled to an albumin binding domain was confirmed using two otherAFFIBODY® molecules: (Z_(Aβ3))₂ and ABD-(Z_(Aβ3))₂.

(Z_(Aβ3))₂ a dimer of a variant of protein Z, in turn derived from the Bdomain of staphylococcal protein A. The Z_(Aβ3) variant was producedusing recombinant DNA technology, through expression of the DNA sequenceencoding it, according to known molecular biology procedures. Used forcomparative purposes.

ABD-(Z_(Aβ3))₂—a fusion protein between the albumin binding domain (ABD)and a dimer of the Z variant Z_(Aβ3) prepared in accordance with knownmolecular biology procedures. Used to illustrate the invention.

His₆-Z_(Taq4:1)—the his-tagged variant of protein Z described inExample 1. Used for comparative purposes.

His₆-Z_(Taq4:5)—the his-tagged variant of protein Z described in Example2. Used for comparative purposes.

Materials and Methods

Mice were injected with two different dimeric AFFIBODY® molecules,(Z_(Aβ3))₂ and ABD-(Z_(Aβ3))₂. Plasma from mice was obtained as before,four times during the treatment scheme and after booster and wasanalyzed for presence of AFFIBODY®-specific IgG. The results showed thatthe mice injected with (Z_(Aβ3))₂, generated a strong IgG response. Theantibody production started around day 14 and peaked at day 34. Theaverage concentration of specific IgG at death bleeding was 278 μg/ml(Z_(Aβ3))₂. No specific antibodies were detected in plasmas from micetreated with ABD-(Z_(Aβ3))₂.

Total IgG-concentration was determined in plasma at day 0 and day 34from both groups of mice. The concentration of total IgG increasedapproximately 2 times in both groups over the 34 day period.

An inhibition-ELISA was set up to test if anti-(Z_(Aβ3))₂-antibodieswere able to neutralize the interaction between the AFFIBODY® moleculeand the target protein. The results showed that (Z_(Aβ3))₂-specific IgGdid not neutralize the interaction between (Z_(Aβ3))₂ and β-Amyloid 40.

Mice and Administration Schedule

The mice were treated with (Z_(Aβ3))₂ and (Z_(Aβ3))₂-ABD according tothe scheme (Table 11). The injection and bleeding scheme is shown inTable 12. Mouse 5 in group 1 became sick and was removed from the studyafter bleeding day 22.

TABLE 11 μg/ ml/ animal/ animal/ Mouse injec- injec- Animal GroupTreatment strain Admin. tion tion number 1 (Z_(Aβ3))₂ NMRI s.c. 20 0.1 1-6 2 ABD- NMRI s.c. 20 0.1 7-12 (Z_(Aβ3))₂

TABLE 12 Day Treatment 0 Preserum (=Sample 1) and Injection 1 3Injection 2 6 Injection 3 7 Sample 2 (day 7) 9 Injection 4 12 Injection5 14 Sample 3 (day 14) 21 Sample 4 (day 21), and Injection 6 34 Sample 5(=death bleeding, day 34)Purification of IgG from Mouse Plasma

Total IgG was purified from pooled plasma day 34 (mouse no 5 day 21)from the mice immunized with (Z_(Aβ3))₂. The plasma pool, 2400 μl, wasdiluted 5 times with PBS-T to a total amount of 12 ml before loading ona Z_(wt)-coupled High-Trap column (L0091-98) previously equilibratedwith PBS-T. The column was washed until absorbance values reached zeroand bound IgG was eluted using an acidic elution buffer (0.2 M Glycine,1 mM EGTA, pH 2.8). For neutralization, 1M Tris base was added to afinal concentration of 50 mM. The buffering capacity was restored byadding 1/10 of the elution volume of 10×PBS (2.68 mM KCl, 1.47 mMKH₂PO₄, 137 mM, NaCl, 8.1 mM Na₂HPO₄, pH 7.4, PBS-Tween (PBS-T), 1×PBSwith 0.05% Tween).

ELISA-plates (96 well, flat bottom, high binding Costar No. 9018) werecoated with the appropriate AFFIBODY® molecule diluted in coating bufferto a final concentration of 2 mg/ml. 100 μl of the coating solution wasadded per well and plates were incubated for 1-3 nights at 4° C. Theplates were then washed manually four times with deionized water andblocked with blocking buffer (0.5% Casein (Sigma) in 1×PBS; 200 μl/well)for 1 to 2 hours. Blocking buffer was removed, and 100 μl of serum wasadded to each well in dilution series. After 1 hour incubation, theplates were washed with the automated ELISA-washer or manually fourtimes with PBS-T, and 100 μl of the second step antibody, HRP-conjugatedgoat anti-mouse IgG diluted 1:2000 in blocking buffer, was added to eachwell. The plates were subsequently incubated for 1 hour. Plates werewashed four times with PBS-T and 100 μl of substrate solution(IMMUNOPURE® TMB) was added to each well followed by incubation in thedark. The colour development was stopped after 15 minutes by theaddition of 100 μl of stop solution 2M H₂SO₄. Plates were read at 450 nmin an ELISA-reader with the use of the Magellan software.

Mouse Anti Z_(Aβ3)-Specific IgG ELISA

Plates were coated with (Z_(Aβ3))₂ 2 μg/ml in coating buffer andincubated overnight at 4° C. After washing, plates were blocked asdescribed above. Plasma from mice immunized with (Z_(Aβ3))₂ or withADB-(Z_(Aβ3))₂ was added in 3-fold dilution series starting from 1/100.After incubation, plates were treated as described above. To measure theconcentration of AFFIBODY®-specific IgG, a standard plasma pool wasused, the Scheele 8 pool. The concentration of anti-Z IgG in this serumpool had been determined on His₆-Z_(Taq4:1)-coated plates to 97 μg/ml.The concentration of anti-His₆-Z_(Taq4:5) and anti-(Z_(Aβ3))₂ IgG inScheele 8 pool was determined to be 291 and 97 μg/ml respectively(L0242-14/16). As a positive control we used plasma from mouse #117(Scheele 7; day 73, in a dilution of 1:10.000, which should give an ODvalue close to the inflection point of the standard curve. Negativecontrol was blocking buffer. The limit of detection was 3 ng/ml.

Total IgG ELISA

Plasmas from day 0 and day 34 (mouse no 5 day 21) from both groups ofmice were analyzed regarding the total amount of IgG. In this assay,ELISA-plates were coated with (Fab)₂ fragments of goat anti-mouse IgG-Fcantibodies (Southern Biotech no. 1031-05 0.5 μg/ml). Plasma or standard(mouse IgG) in 3-fold dilution series starting from 1:10000 and 100ng/ml respectively was added to coated wells. The reaction was developedwith HRP-conjugated (Fab)₂ fragments of goat anti-mouse IgG-fabantibodies (0.2 μg/ml). Substrate solution (ImmunoPure® TMB Pierce no.34021) was added and the colour development was stopped after 10 minutesincubation in dark by the addition of stop solution.

Inhibition ELISA Analysis

Plates were coated with (Z_(Aβ3))₂, 2 μg/ml in coating buffer andincubated overnight in 4° C. After washing plates were blocked asdescribed above. Purified anti-(Z_(Aβ3))₂ IgG was added in 3-folddilution series starting from 1 μg/ml. After 1 h incubation the proteinβ-Amyloid 40, biotinylated, was added to the wells without washing inbetween. The final concentration of β-Amyloid 40 BioSite A2275-74D was10 μg/ml. The plates were washed after one additional hour of incubationand depending on what reaction to analyze, either goat-anti-mouse IgGHRP (Dako P0397 diluted 1/2000) or streptavidin-HRP (diluted 1/5000) wasadded. After the final incubation, plates were washed and developed asdescribed above.

Data Analysis

Magellan (Tecan) was used as ELISA reader software. The results wereexported to Excel for data analysis and presentation. For concentrationdeterminations, the XLfit program, formula “Dose response-one site”,equation 205, was used. Processed data from each day and type ofmeasurement i.e. IgG, concentration etc, were represented by an Excelfile.

Results AFFIBODY®-Specific IgG ELISA Group 1

Plasmas from six mice injected with (Z_(Aβ3))₂ was titrated on(Z_(Aβ3))₂-coated plates. All mice responded by day 14, although mouse 5showed a very low response. The maximal anti-AFFIBODY®-IgG level wasobserved at day 34 (FIG. 15). The average concentration of specific IgGat day 34 was 278 μg/ml (Table 13). Mouse no 5 did not respond well andday 21 is the death bleeding sample. No specific IgG response could bedetected in the serum samples from day 0 i.e. before administration(data not shown).

TABLE 14 Concentration of specific IgG (ND = Not Detectable)Concentration of specific IgG in μg/ml Group 1 day 7 day 14 day 21 day34 mouse 1 ND 3.7 9.8 121.3 mouse 2 ND 13.5 51.3 216.3 mouse 3 ND 19.878.2 530.8 mouse 4 ND 3.4 17.2 113.7 mouse 5 ND ND 7.6 —* mouse 6 ND 8.071.1 407.9 Average ND 9 39 278 St dev 6 29 165 ND: Not detected, valueswere below the detection limit 3 ng/ml. *Mouse 5 became sick and wasremoved from the study day 22.

AFFIBODY®-Specific IgG ELISA Group 2

Plasma from six mice injected with ADB-(Z_(Aβ3))₂ was titrated on(Z_(Aβ3))₂-coated plates. No specific IgG concentration could bedetected in any of the bleeding samples. (FIG. 16).

Total IgG ELISA

Plasma from both groups of mice day 0 and day 34 (mouse no 5 day 21),was titrated on (Z_(Aβ3))₂ coated plates. As shown in Table 15 theamount of total IgG increased over the 34 day period in both groups.However, considering that day 0 levels are surprisingly low, theincrease may reflect the normal expansion in a mouse over thatparticular period in life. Plasma from vehicle mice would have been theappropriate control.

TABLE 15 Total IgG concentrations group 1 Group 1 (Z_(Aβ:3))₂ Group 2ABD-(Z_(Aβ:3))₂ Day Day 34/death Day 34/death Mouse no 0/preplasmableeding Day 0/preplasma bleeding 1 828.9 1724.4 812.9 1850.6 2 984.61948.5 931 2108.9 3 1142.1 2673 437.4 1091.7 4 2074.5 3418.2 900.31180.1 5 1789.2 3936.6 778.6 1839.3 6 829.8 1263.6 1322.9 2573.1

Inhibition ELISA: Target Protein

The likelihood that AFFIBODY®-specific IgG antibodies neutralize/inhibitthe interaction between the target protein and the AFFIBODY® moleculewas explored by an inhibition ELISA. The ELISA-plate was coated with(Z_(Aβ3))₂ as described above. Total IgG, purified from a pool of plasmafrom group 1 day 34, was added. The IgG antibodies were added in 3-folddilutions from 1 μg/ml. The antibodies were allowed to bind for one hourto the coated AFFIBODY® molecule before the target protein β-Amyloid 40was added to a final concentration of 10 μg/ml. The reaction wasdeveloped with streptavidin-HRP to visualize the interaction between(Z_(Aβ3))₂ and the target protein (blue line. The interaction betweenthe IgG-antibodies and the coated AFFIBODY® molecule was visualized withanti-mouse IgG HRP (FIG. 17). Specifically, FIG. 17 shows the results ofinhibition ELISA. Binding of purified (Z_(Aβ3))₂-specific mouseantibodies to coated (Z_(Aβ3))₂ in the presence or absence of β-Amyloid40. Purified IgG and target protein developed, with streptavidin HRP(♦). Purified IgG and target protein, developed with anti-mouse IgG HRP(▪). Purified IgG developed with anti-mouse IgG HRP (▴). The experimentwas repeated twice with the same result.

As control the titrated IgG-antibody was allowed to react with thecoated AFFIBODY® molecule without adding any target protein. FIG. 17also shows almost identical ODvalues for the IgG-detection withanti-mouse IgG HRP with or without the target protein. These resultsindicate that (Z_(Aβ3))₂-specific IgG antibodies do not inhibit theinteraction between (Z_(Aβ3))₂ and β-Amyloid 40.

EXAMPLE 5 Reduction of Immune Response Tested in Additional Mouse Strain

As shown in Example 4 we have previously observed that His₆-Z_(Taq4:1)but not Z_(Taq4:1)-ABD induces an antibody response in out bred NMRImice. In this study, an additional out bred mouse strain (CD1) wastested and the results showed that CD1-mice responded as NMRI mice byproducing specific IgG when administered with His₆-Z_(Taq4:1) but notwith Z_(Taq4:1)-ABD. Thus, the immune unresponsiveness observed uponinjection of ABD-fused AFFIBODY® molecule (Z_(Taq4:1)) seems to be ageneral phenomenon in mice.

The nature of ABD-induced unresponsiveness was further analyzed in NMRImice. Four groups of mice received ten (10) AFFIBODY® injections with achange of injected molecule after the fifth injection (according totreatment scheme 2). The results showed that mice, primed withZ_(Taq4:1)-ABD, produced AFFIBODY specific-IgG after antigen-switch toHis₆-Z_(Taq4:1) (group 4). The antibody production started approximately14 days after the switch to His₆-Z_(Taq4:1), which is the number of daysusually necessary for naïve mice to produce antibodies. Mice that wereprimed with Z_(Taq4:1)-ABD before receiving a mixture of Z_(Taq4:1)-ABDand His₆-Z_(Taq4:1) (group 5) also started to produce specific IgGagainst the AFFIBODY® molecule. The observed specific response in group5 was smaller than seen in group 4 which is most likely due to fact thatgroup 5 received half of the injected dose of His₆-Z_(Taq4:1) ascompared to group 4. Mice in group 6 were primed with His₆-Z_(Taq4:1)before receiving Z_(Taq4:1)-ABD and this treatment resulted in acontinued AFFIBODY®-specific IgG production after the antigen switchalthough the titre decreased by time.

This Scheele 9 study was performed mainly for two reasons: 1) does asecond out bred mouse strain (CD1) respond to the administration ofABD-fused and unfused AFFIBODY® molecules in the same way as NMRI mice(BT1-PAR07, BT11-PAR03), and 2) when Z_(Taq4:1)-ABD is injected andfollowed by His6-Z_(Taq4:1), will the mice generate anAFFIBODY®-specific IgG response or not? The answer to the latterquestion helped us clarify whether the observed unresponsiveness towardsABD-fused AFFIBODY® molecules is an active or passive suppressionprocess. The molecules used in each group of animals are listed in Table16 and the two treatment schemes are presented in Tables 17 and 18. Thesetup of this study is illustrated in FIG. 27A.

TABLE 16 Animal number scheme for the Scheele 9 study; AFFIBODY ® 1:His₆-Z_(Taq4:1); AFFIBODY ® 3: Z_(Taq4:1)-ABD μg/ mL/ animal/ animal/Mouse injec- injec- Animal Group Treatment strain Admin. tion tionnumber 1 Affibody 1 CD1 s.c. 20 0.1 144-148 2 Affibody 3 CD1 s.c. 20 0.1149-153 3 Affibody 1 NMRI s.c. 20 0.1 154-158 4 Affibody NMRI s.c. 200.1 159-163 3 + 1 5 Affibody NMRI s.c. 20 0.1 164-168 3 + 1/3 6 AffibodyNMRI s.c. 20 0.1 169-173 1 + 3

TABLE 17 Treatment scheme 1 for group 1 and 2 Day Treatment 0 Preplasma(=Sample 1) 0 Injection 1 3 Injection 2 6 Injection 3 7 Sample 2 9Injection 4 12 Injection 5 14 Sample 3 21 Sample 4 21 Injection 6 34Death-bleeding (=Sample 5)

TABLE 18 Treatment scheme 2 for group 3, 4, 5, and 6 Day Treatment 0Preplasma (=sample 1) 0 Injection 1 3 Injection 2 6 Injection 3 7 Sample2 9 Injection 4 12 Injection 5 14 Sample 3 21 Sample 4 From now on Ingroup 4, 5, and 6: change of antigen 21 Injection 6 24 Injection 7 27Injection 8 28 Sample 5 30 Injection 9 33 Injection 10 35 Sample 6 45Death-bleeding (=sample 7)

Materials and Methods AFFIBODY® ELISA

96 well, flat bottom, high binding Costar ELISA plates were coated withHis₆-Z_(Taq4:1) diluted in coating buffer (10×coating buffer, 150 mMNa₂CO₃, 350 mM NaHCO₃, pH9.6) to a final concentration of 5 μg/□ml. 100μl of the coating solution was added per well, and plates were incubatedfor 1-4 nights at 4° C. The plates were then washed manually four timeswith deionized water and blocked with blocking buffer (200 μl/well) for1 to 2 hours. Blocking buffer was removed and 100 μl of plasma was addedto each well, diluted from 1:100 in blocking buffer, and then in 3-folddilution series. To measure the concentration of AFFIBODY®-specific IgG,a standard plasma pool was used, the Scheele 6B pool. The concentrationof anti His₆-Z_(Taq4:1) IgG in this plasma pool had been determined to120 μg/ml. As a positive control we used plasma from mouse #118 of theScheele 7 study, day 73, in a dilution of 1:9200, which should give anOD value near the inflection point of the standard curve. Negativecontrol was blocking buffer.

After 2 hours of incubation, the plates were washed four times withPBS-T (Phosphate buffered saline (10×PBS) 2.68 mM KCl, 1.47 mM KH₂PO₄,137 mMNaCl, 8.1 mM Na₂HPO₄, pH 7.4; PBS-Tween (PBS-T), 1×PBS with 0.05%Tween) and 100 μl of a second step antibody, HRP-conjugated goatanti-mouse IgG diluted 1:2000 in blocking buffer (PBS-Tween (PBS-T),1×PBS with 0.05 Tween) was added in each well.

The plates were thereafter incubated for 1 hour. Plates were washed fourtimes with PBS-T and 100 μl of the substrate solution (ImmunoPure® TMB)was added to each well. Plates were incubated in the dark and the colordevelopment was stopped after 15 minutes by the addition of 100 μl ofstop solution (2M H₂SO₄). Plates were read at 450 nm in an ELISA-readerwith the use of the Magellan software.

Total IgG ELISA

For determination of total IgG in plasma, a quantitative ELISA wasperformed. The procedure was the same as for the AFFIBODY® ELISA, withthe following differences:

ELISA-plates were coated with AffiniPure (Fab′)₂ fragment goatanti-mouse IgG Jackson 115-006-008 (Fcγ-fragment specific), in aconcentration of 0.5 μg/ml. Dilutions of the first and second step weredone in PBS-T without casein, and the dilution series of plasma began at1:10.000. Standard IgG was ChromPure mouse IgG Jackson 015-000-003,whole molecule, in a dilution series beginning with 100 ng/ml.Peroxidaseconjugated AffiniPure F(ab′)₂ fragment goat anti-mouse IgG(F(ab′)₂-fragment specific) Jackson 115-036-006 diluted 1:2000 was usedas second step antibody.

Data Analysis

Magellan (Tecan) was used as ELISA reader software. The results wereexported to Excel for data analysis and presentation. For concentrationdeterminations, the XLfit program, formula “Dose response-one site”,equation 205, was used.

Results Treatment Scheme 1

Group 1 and 2 consisted of CD1 mice. The animals were injected witheither His₆-Z_(Taq4:1) (group 1) or Z_(Taq4:1)-ABD (group 2), accordingto treatment scheme 1 (Table 17). Blood samples obtained before, during,and after immunization were analyzed on His₆-Z_(Taq4:1) coated platesfor detection of IgG-antibodies specifically recognizingHis₆-Z_(Taq4:1).

Group 1

The five mice that were injected with His₆-Z_(Taq4:1) developed IgGantibodies as expected. All five mice responded by day 14 showing amaximal anti AFFIBODY®-IgG response at day 34 (FIG. 18). In plasmasamples from day 0 (before administration), no immunological responsecould be seen (data not shown). Table 19 shows the concentration ofspecific and total IgG as calculated with the XLfit program. Averageconcentration of specific IgG at day 34 was 85 mg/ml.

TABLE 19 Concentration of specific and total IgG in group 1Concentration of IgG in μg/ml Specific IgG Total IgG Day 7 Day 14 Day 21Day 34 Day 34 m. 144 ND 7.8 14.2 115.3 3706 m. 145 ND 42.9 70.3 107.93065 m. 146 ND 7.7 16.0 63.5 4306 m. 147 0.3 16.7 49.6 128.9 3612 m. 148ND 8.3 8.0 10.2 2472 average 0.3 8.0 31.6 85.2 3432 (ND = notdetectable)

Group 2

Group 2 consisted of five CD1 mice that were injected withZ_(Taq4:1)-ABD. As shown in FIG. 19. No specific antibodies could bedetected in plasma samples from day 0 (dato not shown) or from any othersampling day. Total IgG concentrations are shown in Table 20.

TABLE 20 Concentration of total IgG in group 2 Concentration of totalIgG in μg/ml Day 34 m. 149 1435 m. 150 794 m. 151 1638 m. 152 1050 m.153 2971 Average 1578

Treatment Scheme 2

Group 3 to 6 consisted of NMRI mice that were injected 10 times. Group 3received His₆-Z_(Taq4:1) at all injection times while the other groupshad an injection scheme with a switch of antigen after the first 5injections (treatment scheme 2, Table 3.3.). Blood samples obtainedbefore, during, and after immunization were analyzed on His₆-Z_(Taq4:1)coated plates for IgG-antibodies directed against His₆-Z_(Taq4:1). Noantibody reactivity was found in any of the pre-plasma samples of group3 to 6 (data not shown).

Group 3

Group 3 was injected with His₆-Z_(Taq4:1) according to treatment scheme2 (Table 18).

Specific antibody production could be observed from day 14. Theconcentration of specific IgG increased over-time to reach a maximum inthe death-bleedings (FIG. 20). These data and the concentration of totalIgG are summarized in table 21. The average concentration of specificIgG at day 45 was 71 μg/ml.

TABLE 21 Concentration of specific and total IgG in group 3Concentration of IgG in μg/ml Specific IgG Total IgG Day Day Day Day DayDay Day 7 14 21 28 35 45 45 m. 154 ND 1.0 4.8 8.3 18.7 32.6 2992 m. 155ND 1.6 4.7 10.3 32.7 65.5 2576 m. 156 ND 4.0 6.2 22.1 68.5 167.5 5703 m.157 ND 2.9 3.1 9.5 13.7 35.0 4088 m. 158 ND 3.3 2.9 8.7 34.1 56.1 2285average ND 2.5 4.3 11.8 33.5 71.3 3529 (ND = not detectable)

Group 4

Group 4 received 5 injections of Z_(Taq4:1)-ABD during the first 3weeks, and then 5 additional injections of His₆-Z_(Taq4:1). The animalsdid not react to the first antigen, Z_(Taq4:1)-ABD, but generatedHis₆-Z_(Taq4:1)-specific antibodies from day 35 and on. Day 35corresponds to 14 days after the switch of injected AFFIBODY® molecule(FIG. 21). These data, and the concentration of total IgG are summarizedin Table 22. The total IgG concentrations were normal except for mouse159 that had an unusually high titre. The average concentration ofspecific IgG at day 45 (death-bleeding and 24 days after the switch) was4 μg/ml.

TABLE 22 Concentration of specific and total IgG in group 4Concentration of IgG in μg/ml Specific IgG Total IgG Day Day Day Day DayDay Day 7 14 21 28 35 45 45 m. 159 ND ND ND ND 0.8 9.6 9497 m. 160 ND NDND ND 0.7 1.0 3172 m. 161 ND ND ND ND 0.7 1.2 2554 m. 162 ND ND ND ND2.5 5.6 3719 m. 163 ND ND ND ND 1.0 no no plasma plasma average ND ND NDND 1.1 4.4 4736 (ND = not detectable)

Group 5

Group 5 received five injections of Z_(Taq4:1)-ABD during the first 3weeks and then another five injections of a mixture of Z_(Taq4:1)-ABDand His₆-Z_(Taq4:1) (10 μg/protein). There was no detectable immuneresponse in the animals upon injection of the first antigen i.e.Z_(Taq4:1)-ABD. After switching to HiS₆-Z_(Taq4:1) injections low levelsof specific antibodies could be detected from day 35 (FIG. 22). Thesedata and the concentration of total IgG are summarized in table 23. Theaverage concentration of specific IgG at day 45 was 0.8 μg/ml. Thereaction is weaker than in group 4 which is most likely due to the lowerdose of His₆-Z_(Taq4:1).

TABLE 23 Concentration of specific and total IgG in group 5Concentration of IgG in μg/ml Specific IgG Total IgG Day Day Day Day DayDay Day 7 14 21 28 35 45 45 m. 164 ND ND ND ND 0.4 1.5 2565 m. 165 ND NDND ND 0.6 0.8 1617 m. 166 ND ND ND ND ND 0.5 1284 m. 167 ND ND ND ND 0.31.1 1988 m. 168 ND ND ND ND ND 0.3 2347 Average ND ND ND ND 0.4 0.8 1864(ND = not detectable)

Group 6

Group 6 received five His₆-Z_(Taq4:1) injections during the first 3weeks followed by 5 injections of Z_(Taq4:1)-ABD. The results areillustrated in FIG. 23. The animal's immune system responded with anormal antibody kinetic i.e. as observed earlier upon injection ofHis₆-Z_(Taq4:1). The specific IgG response reached a maximum at day 28and decreased sometime after the antigen switch. These data and theconcentration of total IgG are summarized in Table 24. The averageconcentration of specific IgG at day 28 was 62 μg/ml, and in thedeath-bleedings on day 45, 42 μg/ml.

TABLE 24 Concentration of specific and total IgG in group 6Concentration of IgG in μg/ml Specific IgG Total IgG Day Day Day Day DayDay Day 7 14 21 28 35 45 45 m. 169 ND 2.3 5.8 14.4 11.6 13.6 3295 m. 170ND 2.2 12.6 82.2 90.7 63.3 2659 m. 171 ND 3.8 18.4 39.4 27.2 25.5 2737m. 172 ND 3.5 9.6 115.0 60.3 56.0 5431 m. 173 ND 8.0 18.7 58.3 32.4 53.32941 Average ND 4.0 13.0 61.9 44.4 42.3 3530 (ND = not detectable)

The results are illustrated in FIG. 27B.

Discussion

There were two main purposes to this study, more precisely a) toinvestigate the immunogenicity of His₆-Z_(Taq4:1) and Z_(Taq4:1)-ABD inan additional out bred mouse strain and b) to determine whether theobserved unresponsiveness of ABD-fused AFFIBODY® molecules is due to anactive suppression of the animals' immune system or is merely a passiveignorance of the same.

The results showed that the out bred CD1 mice responded similar toearlier studied NMRI mice by generating specific antibodies recognizingand binding His₆-Z_(Taq4:1) but not Z_(Taq4:1)-ABD. Thus the ABD-inducedunresponsiveness seems to be a general phenomenon rather than a responsepattern only connected to the NMRI strain.

As mentioned above, the mice in group 3 to 6 were injected withdifferent combinations of ABD-fused and unfused Z_(Taq4:1) to determinewhether the ABD-mediated unresponsiveness is an active or passiveprocess. Mice in group 4 and 5 were injected with five doses ofZ_(Taq4:1)-ABD with the main purpose of inducing unresponsiveness andthen followed by five injections of His₆-Z_(Taq4:1)(group 4) or amixture of His₆-Z_(Taq4:1) and Z_(Taq4:1)-ABD (group 5). Both groupsresponded by producing a His₆-Z_(Taq4:1) specific IgG responsesuggesting that injections with ABD-fused AFFIBODY® molecules do notresult in an active suppression of the protein by the immune system i.e.anergy. The results of this study strongly indicate that ABD-fusedAFFIBODY® molecules, by binding to serum albumin, are ignored by themice' immune system rather than actively suppressed by the same. Micethat first received His₆-Z_(Taq4:1) continued to produceAFFIBODY®-specific IgG after the switch to Z_(Taq4:1)-ABD although thelevels decreased slightly.

EXAMPLE 6 Immunogenicity of Chronically Administered Biologically ActiveMolecules in Rat

In this study, the immune responses generated in rats injected withdifferent AFFIBODY® molecules were analyzed over an extended period.This report covers data up to 96 days after immunization. The moleculesused were (ZAβ3)₂ and ABD-(ZAβ3)₂ as described above in Example 4. Theaim of this study was to 1) analyze the ability of (ZAβ3)₂ to inducespecific immune responses in rats and 2) investigate if ABD-(ZAβ3)₂gives a lower immune response compared to an AFFIBODY® molecule withoutABD. Blood samples obtained before, nine times during the immunizationscheme and after the last injection were analyzed for reactivity. Onplates coated with (ZAβ3)₂ the result showed that administration with(ZAβ3)₂ generated an IgG response that increased over time with a largeindividual variability. In contrast, there were no detectable or verylow specific IgG in serum from all rats injected with ABD-fused(ZAβ3)₂-molecules. In addition, no adverse effects were seen in therats.

Methods General ELISA Method

In general, a volume of 100 μl per well was used for all incubationsteps except for blocking where a volume of 200 μl was used. Plates wereincubated 1 day for coating, 1-2 hours for blocking and plasma, 1 hourfor second step antibody and 10 min for substrate solution. Incubationswere done at room temperature except coating that was incubated at 4° C.Washing was done between all steps unless otherwise stated, using theELISA SkanWasher 300, with 4×350 μl washing buffer (PBS-T) per well.Plates were read at 450 nm in a Tecan ELISA reader with use of theMagellan software. PBS-T buffer was used for all dilutions exceptcoating, where coating buffer was used instead.

Rat Anti (ZAβ3)₂-Specific IgG ELISA

Plates were coated with 2 μg/ml of (ZAβ3)₂, in coating buffer andincubated overnight at 4° C. After washing, plates were blocked asdescribed above. Serum from rabbits injected with (ZAβ3)₂ or ABD-(ZAβ3)₂was added in 3-fold dilution series starting from 1/10. Afterincubation, plates were washed and HRP-conjugated goat anti-rat IgG,Southern Biotechnology 3050-05 diluted 1:6000, was added. After thefinal incubation, plates were washed and developed as described above.

Data Analysis

Magellan2 (Tecan) was used as ELISA reader software. The results wereexported to Excel for data analysis and presentation. For concentrationdeterminations the Xlfit3.0 program, formula “Dose response-one site”,equation 205 was used. Processed data from each day and type ofmeasurement i.e. IgG, concentration etc, are represented by an Excelfile.

Results Treatment and Injection Scheme

Two groups of rats, ten per group, were injected with the same dose ofAFFIBODY® molecules (200 μg/ml), about every 28 days. Serum was drawnbefore (day 0), according to the schedule during the injection schemeand two weeks after the last injection (death-bleeding).

TABLE 25 List of AFFIBODY ® molecules and the corresponding group-numberin the study. μg/animal/ ml/animal/ Test Group Treatment Admin.injection injection tube Rat no. 1 (Zaβ3)2 s.c. 100 0.25 A  1-10 2 ABD-s.c. 100 0.25 B 11-20 (Zaβ3)2

TABLE 26 Injection and bleeding scheme for group 1 and 2. Day Treatment0 Preserum/sample 1, Injection 1(100 μg/animal; tube A- B) 3 Injection 2(100 μg/animal; tube A-B) 6 Injection 3 (100 μg/animal; tube A-B) 7Sample 2 14 Sample 3 21 Sample 4 21 Injection 4 (100 μg/animal; tubeA-B) 36 Sample 5 51 Injection 5 (100 μg/animal; tube A-B) 66 Sample 6 81Injection 6 (100 μg/animal; tube A-B) 96 Sample 7 111 Injection 7 (100μg/animal; tube A-B) 141 Injection 8 (100 μg/animal; tube A-B) 156Sample 8 171 Injection 9 (100 μg/animal; tube A-B) 201 Injection 10 (100μg/animal; tube A-B) 216 Sample 9 231 Injection 11 (100 μg/animal; tubeA-B) 261 Injection 12 (100 μg/animal; tube A-B) 276 Sample 10 291Injection 13 (100 μg/animal; tube A-B) 306 Sample 11Injection with (Zaβ3)₂ and ABD-(Zaβ3)₂

Serum samples from individual rats were titrated in 3-fold dilutionseries on (ZAβ3)₂ coated ELISA-plates and analyzed for the presence ofspecific antibodies, as described in the Methods section.

Titration Curves, (ZAβ3)₂

As shown in FIG. 24 serum from rats of group one, injected with (ZAβ3)₂showed no or low response the first two weeks of immunization. After day14, the antibody titers increased steadily and gave a widely spreadresponse. After 96 days all rats responded although the responsemagnitudes were still different between individual animals.

Titration Curves, ABD-(ZAβ3)₂

As shown in FIG. 25 serum from rats of group two, injected withABD-(ZAβ3)₂, consistently showed no or low antibody responses whentested on (ZAβ3)₂-coated plates during the 96 first days of theinjection scheme.

1-108. (canceled)
 109. A method of reducing or eliminating the immuneresponse elicited upon administration of a biologically active proteinto a mammal, comprising coupling said biologically active protein to atleast one moiety capable of binding to a serum albumin of a mammal,wherein the moiety capable of binding to a serum albumin of a mammal isthe 46 amino acid ABD domain of streptococcal protein G or an albuminbinding derivative thereof having from about 40 to 53 amino acidresidues, to form a molecule which has a binding affinity for the serumalbumin such that the K_(D) of the interaction is less than or equal to10⁻⁷ M, and wherein said molecule is capable of reducing or eliminatingthe immune response elicited upon administration of said biologicallyactive protein to said mammal.
 110. The method of claim 109, in whichthe ABD domain or derivative thereof is arranged to enhance its bindingwith human serum albumin.
 111. The method according to claim 110, inwhich the ABD domain or derivative thereof is capable of interacting atleast one of, and preferably all of, residues Phe-228, Ala-229, Ala-322,Val-325, Phe-326, and Met-329 from human serum albumin so as to enhancebinding of the molecule to albumin.
 112. The method according to claim111, in which the ABD domain or derivative thereof includes an aminoacid residue which forms an interaction with the methionine residue atposition 329 of human serum albumin so as to enhance binding of themolecule to albumin.
 113. The method according to claim 110, in whichthe ABD domain or derivative thereof includes an amino acid residuewhich forms an interaction with helix 7 in the human serum albumindomain IIB so as to enhance binding of the molecule to albumin.
 114. Themethod according to claim 110, in which the ABD domain or derivativethereof includes an amino acid residue which forms an interaction withresidues in human serum albumin domain IIA so as to enhance binding ofthe molecule to albumin.
 115. The method according to claim 110, inwhich the ABD domain or derivative thereof includes an amino acidresidue which forms an interaction with residues between helices 2 and 3of human serum albumin so as to enhance binding of the molecule toalbumin.
 116. The method according to claim 109, wherein the moleculehas a binding affinity for serum albumin of less than or equal to 10⁻⁸M.
 117. The method according to claim 116, wherein the molecule has abinding affinity for serum albumin of less than or equal to 10⁻⁹ M. 118.The method according to claim 117, wherein the molecule has a bindingaffinity for serum albumin of less than or equal to 10⁻¹⁰ M.
 119. Themethod according to claim 118, wherein the molecule has a bindingaffinity for serum albumin of less than or equal to 10⁻¹¹ M.
 120. Themethod according to claim 119, wherein the molecule has a bindingaffinity for serum albumin of less than or equal to 10⁻¹² M.
 121. Themethod according to claim 120, wherein the molecule has a bindingaffinity for serum albumin of less than or equal to 10⁻¹³ M.
 122. Themethod according to claim 121, wherein the molecule has a bindingaffinity for serum albumin of less than or equal to 10⁻¹⁴ M.
 123. Themethod of claim 109, wherein the mammal is a human being.
 124. Themethod according to claim 109, wherein the mammal is a non-human mammal.125. The method according to claim 109, wherein the immune response is ahumoral immune response.
 126. The method according to claim 109, whereinthe biological activity of the biologically active protein comprises anability to interact with a target molecule other than a serum albumin.127. The method according to claim 126, wherein the biological activityof the biologically active protein comprises an ability to block theactivity of the target molecule.
 128. The method according to claim 126,wherein the target molecule is present on the surface of a cell. 129.The method according to claim 128, wherein the cell is a cancerous orprecancerous cell.
 130. The method according to claim 129, wherein thetarget molecule present on the surface of the cell is selected fromHER2, CD4, CD20, CD22, CD74, CEA and EpCAM.
 131. The method according toclaim 126, wherein the target molecule is an enzyme.
 132. The methodaccording to claim 126, wherein the target molecule is selected fromhormone receptors and cytokine receptors.
 133. The method according toclaim 126, wherein the target molecule is a toxin.
 134. The methodaccording to claim 133, in which the toxin is a snake toxin.
 135. Themethod according to claim 109, wherein the biologically active proteinis selected from antibodies, staphylococcal protein A, fibronectin,lipocalin, transferrin, and lectin.
 136. The method according to claim135, wherein the biologically active protein is staphylococcal proteinA.
 137. The method according to claim 136, wherein the biologicallyactive protein comprises the B domain of staphylococcal protein A. 138.The method according to claim 109, wherein the biological activity ofthe biologically active protein comprises an enzymatic activity. 139.The method according to claim 109, wherein the biological activity ofthe biologically active protein comprises a hormone activity.
 140. Themethod according to claim 109, wherein the biological activity of thebiologically active protein comprises a pharmaceutical activity. 141.The method according to claim 109, wherein the biologically activeprotein is selected from growth hormone, ciliary neurotrophic factor,granulocyte-macrophage colony stimulating factor, insulin, interferon β,factor VIII, erythropoietin, GL1P and thrombopoietin.